Process for desilylation of oligonucleotides

ABSTRACT

The present invention relates to processes and reagents for oligonucleotide synthesis and purification. One aspect of the present invention relates to compounds useful for activating phosphoramidites in oligonucleotide synthesis. Another aspect of the present invention relates to a method of preparing oligonucleotides via the phosphoramidite method using an activator of the invention. Another aspect of the present invention relates to sulfur-transfer agents. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to a method of preparing a phosphorothioate by treating a phosphite with a sulfur-transfer reagent of the invention. In a preferred embodiment, the sulfur-transfer agent is a 3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the present invention relates to compounds that scavenge acrylonitrile produced during the deprotection of phosphate groups bearing ethylnitrile protecting groups. In a preferred embodiment, the acrylonitrile scavenger is a polymer-bound thiol. Another aspect of the present invention relates to agents used to oxidize a phosphite to a phosphate. In a preferred embodiment, the oxidizing agent is sodium chlorite, chloroamine, or pyridine-N-oxide. Another aspect of the present invention relates to methods of purifying an oligonucleotide by annealing a first single-stranded oligonucleotide and second single-stranded oligonucleotide to form a double-stranded oligonucleotide; and subjecting the double-stranded oligonucleotide to chromatographic purification. In a preferred embodiment, the chromatographic purification is high-performance liquid chromatography.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/050,633, filed Mar. 18, 2008; which is a continuation of U.S. patentapplication Ser. No. 11/099,430, filed Apr. 5, 2005; which claims thebenefit of priority to U.S. Provisional Patent Application No.60/559,782, filed Apr. 5, 2004; the contents of all of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The study of oligonucleotides is a key area of research for manyacademic and industrial laboratories. See S. Agrawal Trends inBiotechnology 1996, 14, 375-382; J. Man Drug Discovery Today 1996, 1,94-102; and W. Rush Science 1997, 276, 1192-1193. The therapeutic anddiagnostic potential of oligonucleotides has sparked a substantialamount of research activity. One important application ofoligonucleotides is the ability to modulate gene and protein function ina sequence-specific manner. However, many research efforts are hamperedby the small quantities of oligonucleotides that are available forstudy. A method to produce large quantities of oligonucleotide compoundshaving high purity would greatly facilitate oligonucleotide research.Furthermore, it would be highly useful to be able to prepare derivativesof certain oligonucleotides. However, the synthesis of oligonucleotidesand their analogs is often a tedious and costly process.

RNA is generally synthesized and purified by methodologies based on thefollowing steps: phosphoramidite coupling using tetrazole as theactivating agent, oxidation of the phosphorus linker to the diester,deprotection of exocyclic amino protecting groups using NH₄OH, removalof 2′-OH alkylsilyl protecting groups using tetra-n-butylammoniumfluoride (TBAF), and gel purification and analysis of the deprotectedRNA. Examples of chemical synthesis, deprotection, purification andanalysis procedures are provided by Usman et al. in J. Am. Chem. Soc.1987, 109, 7845; Scaringe et al. in Nucleic Acids Res. 1990, 18,5433-5341; Perreault et al. in Biochemistry 1991, 30, 4020-4025; andSlim and Gait in Nucleic Acids Res. 1991, 19, 1183-1188. Odai andcoworkers describe reverse-phase chromatographic purification of RNAfragments used to form a ribozyme. See Odai et al. FEBS Lett. 1990, 267,150-152. Unfortunately, the aforementioned chemical synthesis,deprotection, purification and analysis procedures are time consuming(10-15 min. coupling times), subject to inefficient activation of theRNA amidites by tetrazole, incomplete deprotection of the exocyclicamino protecting groups by NH₄OH, limited by the low capacity of RNApurification using gel electrophoresis, and further limited by lowresolution analysis of the RNA by gel electrophoresis. Therefore, theneed exists for improved synthetic processes for the synthesis ofoligonucleotides.

One important class of oligonucleotide analogues are compounds that havea phosphorothioate in place of the phosphodiester linkage.Phosphorothioate analogues are important compounds in nucleic acidresearch and protein research. For example, phosphorothioate-containingantisense oligonucleotides have been used in vitro and in vivo asinhibitors of gene expression. Site-specific attachment of reportergroups onto the DNA or RNA backbone is facilitated by incorporation ofsingle phosphorothioate linkages. Phosphorothioates have also beenintroduced into oligonucleotides for mechanistic studies on DNA-proteinand RNA-protein interactions, as well as catalytic RNAs.

Introduction of phosphorothioate linkages into oligonucleotides,assembled by solid-phase synthesis, can be achieved using either anH-phosphonate approach or a phosphoramidite approach. The H-phosphonateapproach involves a single sulfur-transfer step, carried out after thedesired sequence has been assembled, to convert all of theinternucleotide linkages to phosphorothioates. Alternatively, thephosphoramidite approach features a choice at each synthetic cycle: astandard oxidation provides the normal phosphodiester internucleotidelinkage, whereas a sulfurization step introduces a phosphorothioate atthat specific position in the sequence. An advantage of usingphosphoramidite chemistry is the capability to control the state of eachlinkage, P═O vs. P═S, in a site-specific manner. The earliest studies tocreate phosphorothioates used elemental sulfur, but the success of thephosphoramidite approach is dependent on the availability andapplication of more efficient, more soluble sulfur-transfer reagentsthat are compatible with automated synthesis. Therefore, the need existsfor novel sulfur-transfer reagents that are compatible with automatedoligonucleotide synthesis.

Another important class of oligonucleotides is double-stranded RNA whichcan be used to initiate a type of gene silencing known as RNAinterference (RNAi). RNA interference is an evolutionarily conservedgene-silencing mechanism, originally discovered in studies of thenematode Caenorhabditis elegans (Lee et al, Cell 75:843 (1993); Reinhartet al., Nature 403:901 (2000)). It is triggered by introducing dsRNAinto cells expressing the appropriate molecular machinery, which thendegrades the corresponding endogenous mRNA. The mechanism involvesconversion of dsRNA into short RNAs that direct ribonucleases tohomologous mRNA targets (summarized, Ruvkun, Science 2294:797 (2001)).This process is related to normal defenses against viruses and themobilization of transposons.

Double-stranded ribonucleic acids (dsRNAs) are naturally rare and havebeen found only in certain microorganisms, such as yeasts or viruses.Recent reports indicate that dsRNAs are involved in phenomena ofregulation of expression, as well as in the initiation of the synthesisof interferon by cells (Declerq et al., Meth. Enzymol. 78:291 (1981);Wu-Li, Biol. Chem. 265:5470 (1990)). In addition, dsRNA has beenreported to have anti-proliferative properties, which makes it possiblealso to envisage therapeutic applications (Aubel et al., Proc. Natl.Acad. Sci., USA 88:906 (1991)). For example, synthetic dsRNA has beenshown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci.USA, 62:357-361 (1969)), is active in the treatment of leukemic mice(Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128 (1969)); andinhibits chemically-induced tumorigenesis in mouse skin (Gelboin et al.,Science 167:205-207 (1970)).

Treatment with dsRNA has become an important method for analyzing genefunctions in invertebrate organisms. For example, Dzitoveva et al.showed for the first time, that RNAi can be induced in adult fruit fliesby injecting dsRNA into the abdomen of anesthetized Drosophila, and thatthis method can also target genes expressed in the central nervoussystem (Mol. Psychiatry. 6(6):665-670 (2001)). Both transgenes andendogenous genes were successfully silenced in adult Drosophila byintra-abdominal injection of their respective dsRNA. Moreover, Elbashiret al., provided evidence that the direction of dsRNA processingdetermines whether sense or antisense target RNA can be cleaved by asmall interfering RNA (siRNA)-protein complex (Genes Dev. 15(2): 188-200(2001)).

Two recent reports reveal that RNAi provides a rapid method to test thefunction of genes in the nematode Caenorhabditis elegans; and most ofthe genes on C. elegans chromosome I and III have now been tested forRNAi phenotypes (Barstead, Curr. Opin. Chem. Biol. 5(1):63-66 (2001);Tavernarakis, Nat. Genet. 24(2):180-183 (2000); Zamore, Nat. Struct.Biol. 8(9):746-750 (2001).). When used as a rapid approach to obtainloss-of-function information, RNAi was used to analyze a random set ofovarian transcripts and have identified 81 genes with essential roles inC. elegans embryogenesis (Piano et al., Curr. Biol. 10(24):1619-1622(2000). RNAi has also been used to disrupt the pupal hemocyte protein ofSarcophaga (Nishikawa et al., Eur. J. Biochem. 268(20):5295-5299(2001)).

Like RNAi in invertebrate animals, post-transcriptional gene silencing(PTGS) in plants is an RNA-degradation mechanism. In plants, this canoccur at both the transcriptional and the post-transcriptional levels;however, in invertebrates only post-transcriptional RNAi has beenreported to date (Bernstein et al., Nature 409(6818):295-296 (2001).Indeed, both involve double-stranded RNA (dsRNA), spread within theorganism from a localized initiating area, to correlate with theaccumulation of small interfering RNA (siRNA) and require putativeRNA-dependent RNA polymerases, RNA helicases and proteins of unknownfunctions containing PAZ and Piwi domains.

Some differences are evident between RNAi and PTGS were reported byVaucheret et al., J. Cell Sci. 114(Pt 17):3083-3091 (2001). First, PTGSin plants requires at least two genes—SGS3 (which encodes a protein ofunknown function containing a coil-coiled domain) and MET1 (whichencodes a DNA-methyltransferase)—that are absent in C. elegans, and thusare not required for RNAi. Second, all of the Arabidopsis mutants thatexhibit impaired PTGS are hyper-susceptible to infection by thecucumovirus CMV, indicating that PTGS participates in a mechanism forplant resistance to viruses. RNAi-mediated oncogene silencing has alsobeen reported to confer resistance to crown gall tumorigenesis (Escobaret al., Proc. Natl. Acad. Sci. USA, 98(23):13437-13442 (2001)).

RNAi is mediated by RNA-induced silencing complex (RISC), asequence-specific, multicomponent nuclease that destroys messenger RNAshomologous to the silencing trigger. RISC is known to contain short RNAs(approximately 22 nucleotides) derived from the double-stranded RNAtrigger, but the protein components of this activity remained unknown.Hammond et al. (Science 293(5532):1146-1150 (August 2001)) reportedbiochemical purification of the RNAi effector nuclease from culturedDrosophila cells, and protein microsequencing of a ribonucleoproteincomplex of the active fraction showed that one constituent of thiscomplex is a member of the Argonaute family of proteins, which areessential for gene silencing in Caenorhabditis elegans, Neurospora, andArabidopsis. This observation suggests links between the geneticanalysis of RNAi from diverse organisms and the biochemical model ofRNAi that is emerging from Drosophila in vitro systems.

Svoboda et al. reported in Development 127(19):4147-4156 (2000) thatRNAi provides a suitable and robust approach to study the function ofdormant maternal mRNAs in mouse oocytes. Mos (originally known as c-mos)and tissue plasminogen activator mRNAs are dormant maternal mRNAs thatare recruited during oocyte maturation, and translation of Mos mRNAresults in the activation of MAP kinase. The dsRNA directed towards Mosor TPA mRNAs in mouse oocytes specifically reduced the targeted mRNA inboth a time- and concentration-dependent manner, and inhibited theappearance of MAP kinase activity. See also, Svoboda et al. Biochem.Biophys. Res. Commun. 287(5):1099-1104 (2001).

The need exists for small interfering RNA (siRNA) conjugates havingimproved pharmacologic properties. In particular, the oligonucleotidesequences have poor serum solubility, poor cellular distribution anduptake, and are rapidly excreted through the kidneys. It is known thatoligonucleotides bearing the native phosphodiester (P═O) backbone aresusceptable to nuclease-mediated degradation. See L. L. Cummins et al.Nucleic Acids Res. 1995, 23, 2019. The stability of oligonucleotides hasbeen increased by converting the P═O linkages to P═S linkages which areless susceptible to degradation by nucleases in vivo. Alternatively, thephosphate group can be converted to a phosphoramidate or alkylphosphonate, both of which are less prone to enzymatic degradation thanthe native phosphate. See Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90,544. Modifications to the sugar groups of the oligonucleotide can conferstability to enzymatic degradation. For example, oligonucleotidescomprising ribonucleic acids are less prone to nucleolytic degradationif the 2′-OH group of the sugar is converted to a methoxyethoxy group.See M. Manoharan ChemBioChem. 2002, 3, 1257 and references therein.

Therefore, the need exists for improved synthetic processes thatfacilitate the synthesis of oligonucleotides. Representative examples ofneeded improvements are better activating agents for phosphoramiditecoupling of nucleotides, better sulfur-transfer reagents for preparingphosphorothioate-containing oligonucleotides, and improved proceduresfor purifying oligonucleotides.

SUMMARY OF THE INVENTION

The present invention relates to processes and reagents foroligonucleotide synthesis and purification. One aspect of the presentinvention relates to compounds useful for activating phosphoramidites inoligonucleotide synthesis. Another aspect of the present inventionrelates to a method of preparing oligonucleotides via thephosphoramidite method using an activator of the invention. Anotheraspect of the present invention relates to sulfur-transfer agents. In apreferred embodiment, the sulfur-transfer agent is a3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the presentinvention relates to a method of preparing a phosphorothioate bytreating a phosphite with a sulfur-transfer reagent of the invention. Ina preferred embodiment, the sulfur-transfer agent is a3-amino-1,2,4-dithiazolidine-5-one. Another aspect of the presentinvention relates to compounds that scavenge acrylonitrile producedduring the deprotection of phosphate groups bearing ethylnitrileprotecting groups. In a preferred embodiment, the acrylonitrilescavenger is a polymer-bound thiol. Another aspect of the presentinvention relates to agents used to oxidize a phosphite to a phosphate.In a preferred embodiment, the oxidizing agent is sodium chlorite,chloroamine, or pyridine-N-oxide. Another aspect of the presentinvention relates to methods of purifying an oligonucleotide byannealing a first single-stranded oligonucleotide and secondsingle-stranded oligonucleotide to form a double-strandedoligonucleotide; and subjecting the double-stranded oligonucleotide tochromatographic purification. In a preferred embodiment, thechromatographic purification is high-performance liquid chromatography.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts activator compounds useful in phosphoramidite-mediatedoligonucleotide synthesis.

FIG. 2 depicts activating agents useful in phosphoramidite-mediatedoligonucleotide synthesis.

FIG. 3 depicts activating agents useful in phosphoramidite-mediatedoligonucleotide synthesis.

FIG. 4 depicts sulfur-transfer agents useful in preparingphosphorothioate linkages in oligonucleotides.

FIG. 5 depicts sulfur-transfer agents useful in preparingphosphorothioate linkages in oligonucleotides.

FIG. 6 depicts the results of the synthesis of 25 and 26 with PADS orEDITH. Note that 25=5′-GsCsGGAUCAAACCUCACCAsAsdTsdT-3′ (SEQ ID NO: 1),26=5′-UsUsGGUGAGGUUUGAUCCGsCsdTsdT-3′ (SEQ ID NO: 2), PADS (fresh)indicates that less than 24 hours hads elapsed since dissolving, PADS(aged) indicates that greater than 48 hours had elapsed sincedissolving, and the term “nd” indicates that the value was notdetermined. The term “PADS” refers to the compound (benzylC(O)S)₂. Theterm “EDITH” refers to 3-ethoxy-1,2,4-dithiazolidine-5-one.

FIG. 7 depicts desilylating reagents and assorted bases used inoligonucleotide synthesis.

FIG. 8 depicts acrylonitrile quenching agents.

FIG. 9 depicts a flow chart for siRNA purification and QC. Note: LC-MSindicates liquid-chromatography mass spectrophotometric analysis; andCGE indicates capillary gel electrophoresis analysis.

FIG. 10 depicts the structure of AL-4112, AL-4180, AL-DP-4014, AL-2200,AL-2201, AL-DP-4127, AL-2299, AL-2300, AL-DP-4139, AL-2281, AL-2282, andAL-DP-4140 (SEQ ID NOS: 3-6).

FIG. 11 depicts the first part of the two-strand approach topurification of AL-DP-4014, the components of which are AL-4112 andAL-4180.

FIG. 12 depicts the second part of the two-strand approach topurification of AL-DP-4014, the components of which are AL-4112 andAL-4180. Note: RP HPLC indicates reverse phase high-performance liquidchromatographic analysis. IEX HPLC indicates ion exchangehigh-performance liquid chromatographic analysis.

FIG. 13 depicts a reverse phase HPLC chromatogram of AL-DP-4014.

FIG. 14 depicts a LC-MS chromatogram of AL-DP-4014.

FIG. 15 depicts a mass spectrum of the peak at 9.913 minutes in the LCchromatogram of AL-DP-4014 shown in FIG. 14.

FIG. 16 depicts a capillary gel electrophoresis chromatogram ofAL-DP-4014.

FIG. 17 depicts a reverse phase HPLC chromatogram of AL-DP-4014.

FIG. 18 depicts an ion exchange chromatogram of AL-DP-4014.

FIG. 19 depicts a LC-MS chromatogram of AL-DP-4127.

FIG. 20 depicts a mass spectrum of the peak at 10.616 minutes in the LCchromatogram of AL-DP-4127 shown in FIG. 19.

FIG. 21 depicts a mass spectrum of the peak at 12.921 minutes in the LCchromatogram of AL-DP-4127 shown in FIG. 19.

FIG. 22 depicts a mass spectrum of the peak at 16.556 minutes in the LCchromatogram of AL-DP-4127 shown in FIG. 19.

FIG. 23 depicts a LC-MS chromatogram of AL-DP-4127.

FIG. 24 depicts a mass spectrum of a minor contaminant which appears asa peak at 13.397 minutes in the LC chromatogram of AL-DP-4127 shown inFIG. 23.

FIG. 25 depicts a mass spectrum of a minor contaminant which appears asa peak at 13.201 minutes in the LC chromatogram of AL-DP-4127 shown inFIG. 23.

FIG. 26 depicts a capillary gel electrophoresis chromatogram ofAL-DP-4127.

FIG. 27 depicts a reverse phase HPLC chromatogram of AL-DP-4127.

FIG. 28 depicts an ion exchange chromatogram of AL-DP-4127.

FIG. 29 depicts a LC-MS chromatogram of AL-DP-4139.

FIG. 30 depicts a mass spectrum of the peak at 13.005 minutes in the LCchromatogram of AL-DP-4139 shown in FIG. 29.

FIG. 31 depicts a capillary gel electrophoresis chromatogram ofAL-DP-4139.

FIG. 32 depicts a reverse phase HPLC chromatogram of AL-DP-4139.

FIG. 33 depicts an ion exchange chromatogram of AL-DP-4139.

FIG. 34 depicts a LC-MS chromatogram of AL-DP-4140.

FIG. 35 depicts a mass spectrum of the peak at 13.965 minutes in the LCchromatogram of AL-DP-4140 shown in FIG. 34.

FIG. 36 depicts a mass spectrum of the peak at 17.696 minutes in the LCchromatogram of AL-DP-4140 shown in FIG. 34.

FIG. 37 depicts a capillary gel electrophoresis chromatogram ofAL-DP-4140.

FIG. 38 depicts a reverse phase HPLC chromatogram of AL-DP-4140.

FIG. 39 depicts an ion exchange chromatogram of AL-DP-4140.

FIG. 40 depicts alternative steps for the two-strand RNA purificationprocedure.

FIG. 41 depicts alternative steps for the two-strand RNA purificationprocedure.

FIG. 42 depicts alternative steps for the two-strand RNA purificationprocedure.

FIG. 43 depicts alternative steps for the two-strand RNA purificationprocedure.

FIG. 44 depicts nucleosides bearing various 2′-protecting groups. Note:The term “B” indicates protected C, G, A, U, or 5-Me-U. The term “X”indicates CN, NO₂, CF₃, SO₂R, or CO₂R. The term “X′” indicates CN, NO₂,CF₃, F, or OMe. The term “Z” indicates H or alkyl. The term “R¹”indicates oxazole, thiazole, or azole.

FIG. 45 depicts nucleosides bearing various 2′-protecting groups whichcan be removed by enzymatic cleavage. Note: The term “B” indicates U,5-Me-U, 5-Me-C, G, or A. The term “X” indicates H, CN, NO₂, CF₃. Theterm “X′” indicates H, CN, NO₂, CF₃, SO₂R, or CO₂R.

FIG. 46 depicts nucleosides bearing various base protecting groupsamenable to the present invention. Note R is H, OMe, F, MOE, or TOM.

FIG. 47 depicts RNA building blocks amenable to the present invention,wherein the nucleoside has a TOM protecting group.

FIG. 48 depicts 5′-silyl protected RNA suitable for the silyldeprotection methods described herein. Note: Base is N-benzoyladenine,N-acetylcytosine, N-isoputyrylguanine, or uracil. R is cyclooctyl forguanosine and uridine. R is cyclododecyl for adenosine and cytidine. SeeScaringe, S. A.; Wincott, F, E. and Caruthers, M. H. J. Am. Chem. Soc.1998, 120, 11820-21.

FIG. 49 depicts a general procedure for solid-phase RNA synthesis.

FIG. 50 depicts sulfur-transfer agents useful in preparingphosphorothioate linkages in oligonucleotides.

FIG. 51 depicts building blocks for conjugation of cholesteryl- andaminoalkyl-hydroxyprolinol at the 5′ and 3′-ends of oligonucleotides. Iand III are for 5′-conjugation, and II and IV are for 3′-conjugation.See Example 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes and reagents foroligonucleotide synthesis and purification. Aspects of the processes andreagents are described in the paragraphs below.

Activators for Phosphoramidite-Mediated Synthesis of Oligonucleotides

The most commonly used process in oligonucleotide synthesis using solidphase chemistry is the phosphoramidite approach. In a typical procedure,a phosphoramidite is reacted with a support-bound nucleotide, oroligonucleotide, in the presence of an activator. The phosphoroamiditecoupling-product is oxidized to afford a protected phosphate. A varietyof different phosphoramidite derivatives are known to be compatible withthis procedure, and the most commonly used activator is 1H-tetrazole.Similar processes have been described using a soluble support. SeeBonora et al. Nucleic Acids Res., 1993, 21, 1213-1217. Thephosphoramidite approach is also widely used in solution phasechemistries for oligonucleotide synthesis. In addition,deoxyribonucleoside phosphoramidite derivatives have been used in thesynthesis of oligonucleotides. See Beaucage et al. Tetrahedron Lett.1981, 22, 1859-1862.

Phosphoramidites derivatives from a variety of nucleosides arecommercially available. 3′-O-phosphoramidites are the most widely usedamidites, but the synthesis of oligonucleotides can involve the use of5′-O- and 2′-O-phosphoramidites. See Wagner et al. Nuclosides &Nucleotides 1997, 17, 1657-1660 and Bhan et al. Nuclosides & Nucleotides1997, 17, 1195-1199. There are also many phosphoramidites available thatare not nucleosides (Cruachem Inc., Dulles, Va.; Clontech, Palo Alto,Calif., Glen Research, Sterling, Va., ChemGenes, Wilmington, Mass.).

Prior to performing the phosphoramidite coupling procedure describedabove, the 3′-OH group of the 5′-O-protected nucleoside has to bephosphityled. Additionally, exocyclic amino groups and other functionalgroups present on nucleobase moieties are normally protected prior tophosphitylation. Traditionally, phosphitylation of nucleosides isperformed by treatment of the protected nucleosides with aphosphitylating reagent such aschloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine which is veryreactive and does not require an activator or2-cyanoethyl-N,N,N′,N′-tetraiso-propylphosphorodiamidite (bis amiditereagent) which requires an activator. After preparation, the nucleoside3′-O-phosphoramidite is coupled to a 5′-OH group of a nucleoside,nucleotide, oligonucleoside or oligonucleotide. The activator mostcommonly used in phosphitylation reactions is 1H-tetrazole.

Despite the common usage of 1H-tetrazole in phosphoramidite coupling andphosphitylation reactions, there are inherent problems with the use of1H-tetrazole, especially when performing larger scale syntheses. Forexample, 1H-tetrazole is known to be explosive. According to thematerial safety data sheet (MSDS) 1H-tetrazole (1H-tetrazole, 98%) canbe harmful if inhaled, ingested or absorbed through the skin. The MSDSalso states that 1H-tetrazole can explode if heated above its meltingtemperature of 155° C. and may form very sensitive explosive metalliccompounds. Hence, 1H-tetrazole requires special handling during itsstorage, use, and disposal.

In addition to its toxicity and explosive nature, 1H-tetrazole is acidicand can cause deblocking of the 5′-O-protecting group and can also causedepurination during the phosphitylation step of amidite synthesis. SeeKrotz et al. Tetrahedron Lett. 1997, 38, 3875-3878. Inadvertentdeblocking of the 5′-O-protecting group is also a problem whenchloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine is used. Recently,trimethylchlorosilane has been used as an activator in thephosphitylation of 5′-O-DMT nucleosides with bis amidite reagent, butthis reagent is usually contaminated with HCl which leads todeprotection and formation of undesired products. See W. Dabkowski etal. Chem. Comm. 1997, 877. The results for this phosphitylation arecomparable to those for 1H-tetrazole. Activators with a higher pKa(i.e., less acidic) than 1H-tetrazole (pKa 4.9) such as4,5-dicyanoimidazole (pKa 5.2) have been used in the phosphitylation of5′-O-DMT thymidine. See C. Vargeese Nucleic Acids Res. 1998, 26,1046-1050.

Another disadvantage to using 1H-tetrazole is the cost of the reagent.The 2003 Aldrich Chemical Company catalog lists 1H-tetrazole at overseven dollars a gram. Furthermore, due to the explosive nature of1H-tetrazole it is only listed as a dilute solution in acetonitrile.This reagent is used in excess of the stoichiometric amount ofnucleoside present in the reaction mixture resulting in considerablecost, especially during large-scale syntheses.

The solubility of 1H-tetrazole is also a factor in the large-scalesynthesis of phosphoramidites, oligonucleotides and their analogs. Thesolubility of 1H-tetrazole is about 0.5 M in acetonitrile. This lowsolubility is a limiting factor on the volume of solvent that isnecessary to run a phosphitylation reaction. An activator having highersolubility would be preferred in order to minimize the volume ofsolvents used in the reactions, thereby lowering the cost and theproduction of waste effluents. Furthermore, commonly used 1H-tetrazole(0.45 M solution) for oligonucleotide synthesis precipitates1H-tetrazole when the room temperature drops below 20° C. Inadvertentprecipitation of 1H-tetrazole can block the lines on an automatedsynthesizer leading to synthesis failure.

In response to the problems associated with the use of 1H-tetrazole,several activators for phosphoramidite coupling have been reported.5-Ethylthio-1H-tetrazole (Wincott, F., et al. Nucleic Acids Res. 1995,23, 2677) and 5-(4-nitrophenyl)-1H-tetrazole (Pon, R. T. TetrahedronLett. 1987, 28, 3643) have been used for the coupling of stericallycrowded ribonucleoside monomers e.g. for RNA-synthesis. The pKa's fortheses activators are 4.28 and 3.7 (1:1 ethanol:water), respectively.The use of pyridine hydrochloride/imidazole (pKa 5.23 (water)) as anactivator for coupling of monomers was demonstrated by the synthesis ofa dimer (Gryaznov, S. M.; Letsinger, L. M. Nucleic Acids Res. 1992, 20,1879). Benzimidazolium triflate (pKa 4.5 (1:1 ethanol:water)) (Hayakawaet al. J. Org. Chem. 1996, 61, 7996-7997) has been used as an activatorfor the synthesis of oligonucleotides having bulky or sterically crowdedphosphorus protecting groups such as aryloxy groups. The use ofimidazolium triflate (pKa 6.9 (water)) was demonstrated for thesynthesis of a dimer in solution (Hayakawa, Y.; Kataoka, M. NucleicAcids and Related Macromolecules: Synthesis, Structure, Function andApplications, Sep. 4-9, 1997, Ulm, Germany). The use of4,5-dicyanoimidazole as an activator for the synthesis of nucleosidephosphoramidite and several 2′-modified oligonucleotides includingphosphorothioates has also been reported.

Due to ongoing clinical demand, the synthesis of oligonucleotides andtheir analogs is being performed on increasingly larger scale reactionsthan in the past. See Crooke et al. Biotechnology and GeneticEngineering Reviews 1998, 15, 121-157. There exists a need forphosphoramidite activators that pose fewer hazards, are less acidic, andless expensive than activating agents that are currently being used,such as 1H-tetrazole. This invention is directed to this, as well asother, important ends.

Activators of the Invention

The activator compounds of the invention have superior properties foractivating phosphoramidites used in oligonucleotide synthesis. Theactivator compounds are generally less explosive and more soluble inacetonitrile than 1H-tetrazole. In addition, the activator compounds ofthe invention required shorter reaction times in the synthesis of adecamer RNA molecule compared to 1H-tetrazole. See Example 1. In certaininstances, the activator compound of the invention has anelectron-withdrawing group to decrease the pKa of the compound. Moreacidic activator compounds can increase the rate of the phosphoramiditecoupling reaction in certain instances. Importantly, shorter reactiontimes minimize the opportunity for side reactions to occur, therebyproviding the desired product in higher purity. In addition, activatorcompounds of the invention can be the free heterocyclic compound or amixture of the activator and its corresponding monoalkyl, dialkyl, ortrialkyl ammonium salt with varying salt to activator molar ratio.Select preferred activator compounds of the invention are presented inFIGS. 1, 2, and 3.

One aspect of the present invention relates to a compound represented byformula I:

wherein

X is C(R⁶) or N;

R¹, R², R³, and R⁶ each independently represent H, —NO₂, —CN, —CF₃,—SO₂R⁸, —SR⁸, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxyl,—OR⁷, —N(R⁷)₂, —N(R⁷)C(O)R⁸, —C(O)R⁷, or —CO₂R⁸; or an instance of R¹and R², or R² and R³ can be taken together to form a 4-8 member ringcontaining 0-4 heteratoms selected from the group consisting of O, N andS;

R⁴ is absent or represents independently for each occurrence—(C(R⁹)₂)_(n)CH₃.Y;

R⁵ is H or —(C(R⁹)₂)_(n)CH₃;

R⁷ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁸ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁹ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁸CO₂ ⁻.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is C(R⁶).

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is N.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is C(R⁶); R¹, R², R³, and R⁶ eachindependently represent H, —NO₂, or —CN; R⁴ is absent; and R⁵ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is C(R⁶); R¹, R², R³, and R⁶ are H;R⁴ is absent; and R⁵ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is N; R¹, R², and R³ are H; R⁴ isabsent; and R⁵ is H.

Another aspect of the present invention relates to a compoundrepresented by formula II:

wherein

R¹ and R³ each represent independently H, —NO₂, —CN, —CF₃, —SO₂R⁶, —SR⁶,halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R⁵)C(O)R⁶, —C(O)R⁵,or —CO₂R⁶;

R² is absent or represents independently for each occurrence—(C(R⁷)₂)_(n)CH₃.Y;

R⁴ is H or —(C(R⁷)₂)_(n)CH₃;

R⁵ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁶ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁷ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁶CO₂ ⁻.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ and R³ each represent independentlyH, —NO₂, or —CN; R² is absent; and R⁴ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is H; R³ is —NO₂; R² is absent; andR⁴ is H.

Another aspect of the present invention relates to a compoundrepresented by formula II:

wherein

R¹ and R² each represent independently H, —NO₂, —CN, —CF₃, —SO₂R⁶, —SR⁶,halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R⁵)C(O)R⁶, —C(O)R⁵,or —CO₂R⁶;

R³ is absent or represents independently for each occurrence—(C(R⁷)₂)_(n)CH₃.Y;

R⁴ is H or —(C(R⁷)₂)_(n)CH₃;

R⁵ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁶ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁷ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁶CO₂ ⁻.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ and R² each represent independentlyH, —NO₂, or —CN; R⁴ is absent; and R⁴ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is H; R² is —NO₂; R³ is absent; andR⁴ is H.

Another aspect of the present invention relates to a compoundrepresented by formula IV:

wherein

R¹ is H, —SR⁵, alkyl, aryl, —N(R⁴)₂, —(C(R⁴)₂)_(m)CO₂R⁵, —NO₂, —CN,—CF₃, —SO₂R⁵, —SR⁵, halogen, alkenyl, alkynyl, aralkyl, —N(R⁴)C(O)R⁵,—C(O)R⁴, or —CO₂R⁵;

R² is absent or represents independently for each occurrence—(C(R⁶)₂)_(n)CH₃.Y;

R³ is H or —(C(R⁶)₂)_(n)CH₃;

R⁴ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁵ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁶ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive;

m is 1, 2, 3, 4, 5, 6, 7, or 8; and

Y represents independently for each occurrence halogen or R⁵CO₂ ⁻.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is —SR⁵, alkyl, aryl, —N(R⁴)₂, or—(C(R⁴)₂)_(m)CO₂R⁵.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is absent, and R³ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is —SR⁵, alkyl, aryl, —N(R⁴)₂, or—(C(R⁴)₂)_(m)CO₂R⁵; R² is absent; R³ is H; R⁴ is H; R⁵ is alkyl oraralkyl; and m is 1.

Another aspect of the present invention relates to a compoundrepresented by formula V:

wherein

R¹, R³, and R⁴ each represent independently H, —NO₂, —CN, —CF₃, —SO₂R⁷,—SR⁷, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R⁶)C(O)R⁵,—C(O)R⁶, or —CO₂R⁷;

R² is absent or represents independently for each occurrence—(C(R⁸)₂)_(n)CH₃.Y;

R⁵ is H or —(C(R⁸)₂)_(n)CH₃;

R⁶ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁷ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁸ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁷CO₂ ⁻.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is absent, and R⁵ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ is H, R² is absent, R³ and R⁴ are—CN, and R⁵ is H.

Another aspect of the present invention relates to a method of forming aphosphite compound, comprising the steps of:

admixing a phosphoramidite, alcohol, and activating agent to form aphosphite compound, wherein said activating agent is selected from thegroup consisting of

wherein

X is C(R⁶) or N;

R¹, R², R³, and R⁶ each independently represent H, —NO₂, —CN, —CF₃,—SO₂R⁸, —SR⁸, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkoxyl,—OR⁷, —N(R⁷)₂, —N(R⁷)C(O)R⁸, —C(O)R⁷, or —CO₂R⁸; or an instance of R¹and R⁶, R¹ and R², or R² and R³ can be taken together for form a 4-8member ring containing 0-4 heteratoms selected from the group consistingof O, N and S;

R⁴ is absent or represents independently for each occurrence—(C(R⁹)₂)_(n)CH₃.Y;

R⁵ is H or —(C(R⁹)₂)_(n)CH₃;

R⁷ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁸ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁹ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁸CO₂ ⁻;

wherein

R¹ and R³ each represent independently H, —NO₂, —CN, —CF₃, —SO₂R⁶, —SR⁶,halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R⁵)C(O)R⁶, —C(O)R⁵,or —CO₂R⁶;

R² is absent or represents independently for each occurrence—(C(R⁷)₂)_(n)CH₃.Y;

R⁴ is H or —(C(R⁷)₂)_(n)CH₃;

R⁵ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁶ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁷ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁶CO₂ ⁻;

wherein

R¹ and R² each represent independently H, —NO₂, —CN, —CF₃, —SO₂R⁶, —SR⁶,halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R⁵)C(O)R⁶, —C(O)R⁵,or —CO₂R⁶;

R³ is absent or represents independently for each occurrence—(C(R⁷)₂)_(n)CH₃.Y;

R⁴ is H or —(C(R⁷)₂)_(n)CH₃;

R⁵ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁶ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁷ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁶CO₂ ⁻;

wherein

R¹ is H, —SR⁵, alkyl, aryl, —N(R⁴)₂, —(C(R⁴)₂)_(m)CO₂R⁵, —NO₂, —CN,—CF₃, —SO₂R⁵, —SR⁵, halogen, alkenyl, alkynyl, aralkyl, —N(R⁴)C(O)R⁵,—C(O)R⁴, or —CO₂R⁵;

R² is absent or represents independently for each occurrence—(C(R⁶)₂)_(n)CH₃.Y;

R³ is H or —(C(R⁶)₂)_(n)CH₃;

R⁴ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁵ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁶ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive;

m is 1, 2, 3, 4, 5, 6, 7, or 8; and

Y represents independently for each occurrence halogen or R⁵CO₂ ⁻; and

wherein

R¹, R³, and R⁴ each represent independently H, —NO₂, —CN, —CF₃, —SO₂R⁷,—SR⁷, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, —N(R⁶)C(O)R⁵,—C(O)R⁶, or —CO₂R⁷;

R² is absent or represents independently for each occurrence—(C(R⁸)₂)_(n)CH₃.Y;

R⁵ is H or —(C(R⁸)₂)_(n)CH₃;

R⁶ represents independently for each occurrence H, alkyl, aryl, oraralkyl;

R⁷ represents independently for each occurrence alkyl, aryl, or aralkyl;

R⁸ represents independently for each occurrence H or alkyl;

n represents independently for each occurrence 0 to 15 inclusive; and

Y represents independently for each occurrence halogen or R⁷CO₂ ⁻.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphoramidite is a 3′-nucleosidephosphoramidite, 3′-nucleotide phosphoramidite, or 3′-oligonucleotidephosphoramidite.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphoramidite is represented byformula A:

wherein

R₁ is alkyl, aryl, aralkyl, or —Si(R₅)₃; wherein said alkyl, aryl, andaralkyl group is optionally substituted with —CN, —NO₂, —CF₃, halogen,—O₂CR₅, or —OSO₂R₅;

R₂ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, or alkenyl;

R₃ and R₄ each represent independently alkyl, cycloalkyl,heterocycloalkyl, aryl, or aralkyl; or R₃ and R₄ taken together form a3-8 member ring; and

R₅ is alkyl, cycloalkyl, heterocycloalkyl, aryl, or aralkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is —CH₂CH₂CN.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substitutedheterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is a nucleoside or nucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₃ and R₄ are alkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is an optionally substitutedribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein alcohol is a nucleoside, nucleotide, oroligonucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is represented by R₅—OH,wherein R₅ is optionally substituted alkyl, cycloalkyl,heterocycloalkyl, aryl, aralkyl, alkenyl, or—(C(R₆)₂)_(p)heterocycloalkyl; R₆ is H or alkyl; and p is 1, 2, 3, 4, 5,6, 7, or 8.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₅ is —(C(R₆)₂)_(p)heterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, further comprising the step of admixing aproton-shuttle compound to the mixture comprising said phosphoramidite,said alcohol, and said activating agent, wherein the pKa of saidproton-shuttle compound is greater than the pKa of said activatingagent, and the pKa of said proton-shuttle compound is less than the pKaof said phosphoramidite.

In certain embodiments, the present invention relates to theaforementioned method, wherein said proton-shuttle compound is aprimary, secondary, or tertiary amine.

In certain embodiments, the present invention relates to theaforementioned method, wherein said proton-shuttle compound isrepresented by N(R₇)(R₈)R₉, wherein R₇, R₈, and R₉ each representindependently for each occurrence H, alkyl, cycloalkyl, aryl, aralkyl,alkenyl; or R₇ and R₈ taken together form a 3-8 membered ring; and R₉ isH, alkyl, cycloalkyl, aryl, or aralkyl.

Sulfur-Transfer Reagents

Modified oligonucleotides are of great value in molecular biologicalresearch and in applications such as anti-viral therapy. Modifiedoligonucleotides which can block RNA translation, and are nucleaseresistant, are useful as antisense reagents. Sulfurized oligonucleotidescontaining phosphorothioate (P═S) linkages are of interest in theseareas. Phosphorothioate-containing oligonucleotides are also useful indetermining the stereochemical pathways of certain enzymes whichrecognize nucleic acids.

Standard techniques for sulfurization of phosphorus-containing compoundshave been applied to the synthesis of sulfurized deoxyribonucleotides.Examples of sulfurization reagents which have been used includeelemental sulfur, dibenzoyl tetrasulfide, 3-H-1,2-benzidithiol-3-one1,1-dioxide (also known as Beaucage reagent), tetraethylthiuramdisulfide (TETD), and bis(O,O-diisopropoxy phosphinothioyl) disulfide(known as Stec reagent). Most of the known sulfurization reagents,however, have one or more significant disadvantages.

Elemental sulfur presents problems and is not suitable for automationbecause of its insolubility in most organic solvents. Furthermore,carbon disulfide, a preferred source of sulfur, has undesirablevolatility and an undesirably low flash point. Unwanted side productsare often observed with the use of dibenzoyl tetrasulfide. The Beaucagereagent, while a relatively efficient sulfurization reagent, isdifficult to synthesize and not particularly stable. Furthermore, use ofBeaucage reagent forms a secondary reaction product which is a potentoxidizing agent. See R. P. Iyer et al. J. Am. Chem. Soc. 1990, 112,1253-1254 and R. P. Iyer et al. J. Org. Chem. 1990, 55, 4693-4699. Thiscan lead to unwanted side products which can be difficult to separatefrom the desired reaction product. Tetraethylthiuram disulfide, whilerelatively inexpensive and stable, has a sulfurization reaction ratewhich can be undesirable slow.

A method for producing a phosphorothioate ester by reaction of aphosphite ester with an acyl disulfide is disclosed in Dutch patentapplication No. 8902521. The disclosed method is applied to a purifiedphosphotriester dimer utilizing solution-phase chemistry. The method istime and labor intensive in that it was only shown to work in a complexscheme which involved carrying out the first stage of synthesis(formation of a phosphite) in acetonitrile, removing the acetonitrile,purifying the intermediate phosphotriester, and proceeding with thesulfurization in a solvent mixture of dichloroethane (DCE) and2,4,6-collidine. Furthermore, the method was demonstrated only with adinucleotide. There was no suggestion that the Dutch method could beemployed with larger nucleic acid structures, that the same could employa common solvent throughout all steps of synthesis, that improved yieldscould be obtained, or that the method could be adapted for conventionalautomated synthesis without extensive modification of the scheme ofautomation. Although acetonitrile is mentioned as one of severalpossible solvents, utility of the method for carrying out all steps ofthe synthesis in acetonitrile as a common solvent was not demonstrated.While other publications (Kamer et al. Tetrahedron Lett. 1989, 30(48),6757-6760 and Roelen et al. Rech. Tray. Chim. Pays-Bas 1991, 110,325-331) show sulfurization of oligomers having up to six nucleotides,the aforementioned shortcomings are not overcome by the methodsdisclosed in these references.

A thioanhydride derivative EDITH (3-ethoxy-1,2,4-dithiazolidine-5-one)is disclosed in U.S. Pat. No. 5,852,168 (the '168 application). Hereinwe have established that, contrary to expectations, this reagent can beused in the synthesis of 2′-substituted RNA and chimeric RNA.Importantly, even though these reaction conditions are basic they do notresult in elimination of the 2′-substitutent or other degredation of theRNA.

Finally, PADS (phenylacetyl disulfide) is disclosed in U.S. Pat. Nos.6,242,591 and 6,114,519. These patents disclose a methof ofsulfurization carried out by contacting a deoxynucleic acid with anacetyl disulfide for a time suffiient to effect formation of aphosphorothioate functional group. However, these patents do not provideexamples of such a reaction in the syntheis of RNA (including2′-substituted RNA and chimeric RNA), as is demonstrated herein. Inaddition, even though these reaction conditions are basic they do notresult in elimination of the 2′-substitutent or other degredation of theRNA.

Thus, the need exists for improved methods and reagents for preparingsulfur-containing phosphorous groups, such as phosphorothioate linkages,in oligonucleotides and other organic compounds. The present inventionrelates to sulfur-transfer reagents and methods for the formation ofphosphorothioates. The methods are amenable to the formation ofphosphorothioate linkages in oligonucleotides or derivatives, withoutthe need for complex solvent mixtures, repeated washing, or solventchanges.

Certain preferred sulfur-transfer reagents of the invention arepresented in FIGS. 4, 5, and 50.

One aspect of the present invention relates to the compound representedby formula D:

wherein

X represents independently for each occurrence C(O), C(S), SO₂, CO₂,CS₂, or SO_(;)

R¹ and R² represent independently for each occurrence alkyl, cycloalkyl,aryl, heteroaryl; aralkyl, heteroaralkyl, or —N(R³)R⁴; or R¹ and R²taken together form an optionally substituted aromatic ring;

R³ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁴ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

n is 2, 3, or 4; and

provided that when X is C(O), R¹ is not benzyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 2.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R¹ and R² are phenyl, benzyl,cyclohexyl, pyrrole, pyridine, or —CH₂-pyridine.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is C(O), R¹ is phenyl, and R² isphenyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is SO₂, R¹ is phenyl, and R² isphenyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is C(O), R¹ is pyrrole, and R² ispyrrole.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is C(O), and R¹ and R² taken togetherform a phenyl ring.

Another aspect of the present invention relates to the compoundrepresented by formula D1:

wherein

X is CN, P(OR²)₂, P(O)(OR²)₂, C(O)R¹, C(S)R¹, SO₂R¹, CO₂R¹, CS₂R¹, orSOR¹;

Y is CN, P(OR²)₂, or P(O)(OR²)₂;

R¹ represents independently for each occurrence alkyl, cycloalkyl, aryl,heteroaryl; aralkyl, heteroaralkyl, or —N(R³)R⁴;

R² represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl; aralkyl, heteroaralkyl, alkali metal, or transitionmetal; or two instances of R² taken together form an alkaline earthmetal or transitional metal with an overall charge of +2.

R³ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁴ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;and

n is 2, 3, or 4.

In certain embodiments, the present invention relates to theaforementioned compound, wherein n is 2.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is CN.

In certain embodiments, the present invention relates to theaforementioned compound, wherein Y is P(OR²)₂.

Another aspect of the present invention relates to the compoundrepresented by formula E:

wherein

X is O or S;

R¹ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R² is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,—C(O)N(R³)R⁴, —C(S)N(R³)R⁴, —C(S)N(R³)₂, —C(S)OR⁴, —CO₂R⁴, —C(O)R⁴, or—C(S)R⁴;

R³ is H or alkyl; and

R⁴ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is O.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is H, alkyl, or cycloalkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is aryl or aralkyl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R² is —C(O)N(R³)R⁴, —C(S)N(R³)R⁴,—C(S)N(R³)₂, —C(S)OR⁴, —CO₂R⁴, —C(O)R⁴, or —C(S)R⁴.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R³ is H.

In certain embodiments, the present invention relates to theaforementioned compound, wherein R⁴ is alkyl or aryl.

In certain embodiments, the present invention relates to theaforementioned compound, wherein X is O, and R² is H.

Another aspect of the present invention relates to a compound formed bythe process, comprising the steps of:

admixing about 1 equivalent of chlorocarbonyl sulfenyl chloride, about 1equivalent of thiourea, and about 1 equivalent of triethylamine in acontainer cooled with a ice-bath at about 0° C. under an atmosphere ofargon, stirring the resultant mixture for about 6 hours, filtering saidmixture, concentrating said mixture to give a residue, andrecrystallizing said residue from dichloromethane-hexanes to give thecompound.

Another aspect of the present invention relates to a method of forming aphosphorothioate compound, comprising the steps of:

admixing a phosphite and a sulfur transfer reagent to form aphosphorothioate, wherein said sulfur transfer reagent is selected fromthe group consisting of MoS₄.Et₃NCH₂Ph,

wherein

X represents independently for each occurrence C(O), C(S), SO₂, CO₂,CS₂, or SO_(;)

R¹ and R² represent independently for each occurrence alkyl, cycloalkyl,aryl, heteroaryl; aralkyl, heteroaralkyl, or —N(R³)R⁴; or R¹ and R²taken together form an optionally substituted aromatic ring;

R³ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁴ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

n is 2, 3, or 4; and

provided that when X is C(O), R¹ is not benzyl;

wherein

X is CN, P(OR²)₂, P(O)(OR²)₂, C(O)R¹, C(S)R¹, SO₂R¹, CO₂R¹, CS₂R¹, orSOR¹;

Y is CN, P(OR²)₂, or P(O)(OR²)₂;

R¹ represents independently for each occurrence alkyl, cycloalkyl, aryl,heteroaryl; aralkyl, heteroaralkyl, or —N(R³)R⁴;

R² represents independently for each occurrence H, alkyl, cycloalkyl,aryl, heteroaryl; aralkyl, heteroaralkyl, alkali metal, or transitionmetal; or two instances of R² taken together form an alkaline earthmetal or transitional metal with an overall charge of +2.

R³ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R⁴ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;and

n is 2, 3, or 4; and

wherein

X is O or S;

R¹ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;

R² is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,—C(O)N(R³)R⁴, —C(S)N(R³)R⁴, —C(S)N(R³)₂, —C(S)OR⁴, —CO₂R⁴, —C(O)R⁴, or—C(S)R⁴;

R³ is H or alkyl; and

R⁴ is H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphite is represented by formulaF:

wherein

R₁ is alkyl, aryl, aralkyl, or —Si(R₄)₃; wherein said alkyl, aryl, andaralkyl group is optionally substituted with —CN, —NO₂, —CF₃, halogen,—O₂CR₅, or —OSO₂R₄;

R₂ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, or alkenyl;

R₃ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, alkenyl, or —(C(R₅)₂)_(p)heterocycloalkyl;

R₄ is alkyl, cycloalkyl, heterocycloalkyl, aryl, or aralkyl;

R₅ is H or alkyl; and

p is 1, 2, 3, 4, 5, 6, 7, or 8.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is —CH₂CH₂CN.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substitutedheterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is a nucleoside, nucleotide, oroligonucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is

wherein R′₁ represents independently for each occurrence alkyl, aryl,aralkyl, or —Si(R₄)₃; wherein said alkyl, aryl, and aralkyl group isoptionally substituted with —CN, —NO₂, —CF₃, or halogen; and n¹ is 1 to50 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 25 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 15 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 10 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 5 inclusive.

Acrylonitrile Quenching Agents

Ethylnitrile is a common phosphate protecting group used inoligonucleotide synthesis. One of the advantages of this protectinggroup is that it can be easily removed by treating the protectedphosphate with a base. The overall transformation is illustrated below.

However, the acrylonitrile generated from the deprotection reaction is agood electrophile which can react with nucleophilic functional groups onthe desired nucleotide or oligonucleotide product. This side-reactionreduces the yield of the desired product and introduces impurities whichcan be difficult to remove. Therefore, the need exists for a reagentthat will react selectively with the acrylonitrile produced during thedeprotection reaction. Representative examples of compounds that wouldserve as acrylonitrile scavenging agents during the deprotectionreaction are polymer-bound thiols, alkane thiol having at least 10carbon atoms, heteroarylthiol, the sodium salt of an alkane thiol, andthiols that have sufficiently low volitility so that they are odorless,e.g., thiols that have a high molecular weight.

Odorless thiols have been described by K. Nishide and M. Node in GreenChem. 2004, 6, 142. Some examples of odorless thiols includedodecanethiol, 4-n-heptylphenylmethanethiol,4-trimethylsilylphenylmethanethiol, and 4-trimethylsilylbenzenethiol.For additional examples see Development of Odorless Thiols and Sulfidesand Their Applications to Organic Synthesis. Nishide, Kiyoharu; Ohsugi,Shin-ichi; Miyamoto, Tetsuo; Kumar, Kamal; Node, Manabu. KyotoPharmaceutical University, Misasagi, Yamashina, Kyoto, Japan.Monatshefte fuer Chemie 2004, 135(2), 189-200. Benzene thiol and benzylmercaptan derivatives having only faint odors have been described byNishide and coworkers. Representative examples include: 4-RC₆H₄X,3-RC₆H₄X and 2-C₆H₄X (R=Me₃Si, Et₃Si or Pr₃Si; X=SH or CH₂SH) SeeNishide, Kiyoharu; Miyamoto, Tetsuo; Kumar, Kamal; Ohsugi, Shin-ichi;Node, Manabu of Kyoto Pharmaceutical University, Misasagi, Yamashina,Kyoto, Japan. in “Synthetic Equivalents of Benzenethiol and BenzylMercaptan Having Faint Smell: Odor Reducing Effect of TrialkylsilylGroup.” Tetrahedron Lett. 2002, 43(47), 8569-8573. See Node andcoworkers for a description of odorless 1-dodecanethiol. andp-heptylphenylmethanethiol. Node, Manabu; Kumar, Kamal; Nishide,Kiyoharu; Ohsugi, Shin-ichi; Miyamoto, Tetsuo. of Kyoto PharmaceuticalUniversity, Yamashina, Misasagi, Kyoto, Japan. in “Odorless substitutesfor foul-smelling thiols: syntheses and applications.” Tetrahedron Lett.2001, 42(52), 9207-9210.

Representative examples of acrylonitrile quenching agents are shown inFIG. 8.

One aspect of the present invention relates to a method of removing anethylcyanide protecting group, comprising the steps of:

admixing a phosphate compound bearing a ethylcyanide group with a basein the presence acrylonitrile scavenger, wherein said acrylonitrilescavenger is a polymer-bound thiol, 4-n-heptylphenylmethanethiol, alkanethiol having at least 10 carbon atoms, heteroarylthiol, the sodium saltof an alkyl thiol,

wherein R¹ is alkyl; and R² is —SH, or —CH₂SH.

In certain embodiments, the present invention relates to theaforementioned method, wherein said acrylonitrile scavenger is

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphate compound is anoligonucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphate compound is anoligonucleotide containing at least one phosphorothioate group.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphate compound is an oligomer ofribonucleotides.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphate is represented by formulaG:

wherein

R₁ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, or alkenyl;

R₂ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, alkenyl, or —(C(R₃)₂)_(p)heterocycloalkyl;

R₃ is H or alkyl; and

p is 1, 2, 3, 4, 5, 6, 7, or 8.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substitutedheterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is a nucleoside, nucleotide, oroligonucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is

wherein R′₁ represents independently for each occurrence alkyl, aryl,aralkyl, or —Si(R₄)₃; wherein said alkyl, aryl, and aralkyl group isoptionally substituted with —CN, —NO₂, —CF₃, or halogen; R₄ is alkyl,aryl, or aralkyl; and n¹ is 1 to 50 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 25 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 15 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 10 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 5 inclusive.

Methods for Preserving P═S Bonds

The P═S bond of phosphorothioate nucleotides is sensitive to oxidizingagents, resulting in conversion of the P═S bond to a P═O bond. Oneaspect of the present invention relates to methods of preventingunwanted oxidation of the P═S bond. One method of preventing unwantedoxidation of the P═S bond is to mix a compound which is more readilyoxidized than the P═S bond of a phosphothioate group with thephosphorothioate-containing nucleotide. Examples of compounds that areoxidized more readily than the P═S bond of a phosphothioate groupinclude 2-hydroxylethanethiol, EDTA, vitamin E, thiols includingodorless thiols, and vitamin C. Other such compounds can be readilyidentified by one of ordinary skill in the art by comparing theoxidation potential of the P═S bond of a phosphorothioate to theantioxidant additive. The antioxidant should be oxidized more easilythan the P═S bond of the phosphorothioate.

Oxidizing Agents for Preparing P═O Bonds

As described above, oligonucleotides having a phosphorothioate linkageare promising therapeutic agents. In certain instances, it isadvantageous to prepare an oligonucleotide having a mixture of phosphateand phosphorothioate linkages. One procedure to prepare oligonucleotideshaving a mixture of phosphate and phosphorothioate linkages involvesattaching a first oligonucleotide to a second oligonucleotide, whereinthe first oligonucleotide consists of nucleosides linked viaphosphorothioate groups, and the second oligonucleotide consists ofnucleosides linked by phosphite groups. Then, the phosphite groups areoxidized to give the phosphate linkage. Alternatively, oligonucleotidescan be added sequentially to the first oligonucleotide using thephosphoramide method. Then, the newly added nucleosides, which arelinked via phosphite groups, are oxidized to convert the phosphitelinkage to a phosphate linkage. One of the most commonly used oxidizingagents for converting a phosphite to a phosphate is I₂/amine.Consequently, the I₂/amine reagent is a very strong oxidant which alsooxidizes phosphorothioates to phosphates. Hence, milder oxidizing agentsare needed which will oxidize a phosphite to a phosphate, but will notoxidize a phosphorothioate group. Three examples of oxidizing agentsthat will oxidize a phosphite to a phosphate, but will not oxidize aphosphorothioate group, are NaClO₂, chloroamine, and pyridine-N-oxide.Additional oxidizing agents amenable to the present invention are CCl₄,CCl₄/water/acetonitrile, CCl₄/water/pyridine, dimethyl carbonate,mixture of KNO₃/TMSCl in CH₂Cl₂, NBS, NCS, or a combination of oxidizingagent, an aprotic organic solvent, a base and water.

One aspect of the present invention relates to a method of oxidizing aphosphite to a phosphate, comprising the steps of:

admixing a phosphite with an oxidizing agent to produce a phosphate,wherein said oxidizing agent is NaClO₂, chloroamine, pyridine-N-oxide,CCl₄, CCl₄/water/acetonitrile, CCl₄/water/pyridine, dimethyl carbonate,mixture of KNO₃/TMSCl in CH₂Cl₂, NBS, or NCS.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oxidizing agent is NaClO₂,chloroamine, or pyridine-N-oxide.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphite is an oligomer of anucleoside linked via phosphite groups.

In certain embodiments, the present invention relates to theaforementioned method, wherein said nucleoside is a ribonucleoside.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphite is represented by formulaH:

wherein

R₁ is alkyl, aryl, aralkyl, or —Si(R₄)₃; wherein said alkyl, aryl, andaralkyl group is optionally substituted with —CN, —NO₂, —CF₃, halogen,—O₂CR₅, or —OSO₂R₄;

R₂ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, or alkenyl;

R₃ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, alkenyl, or —(C(R₅)₂)_(p)heterocycloalkyl;

R₄ is alkyl, cycloalkyl, heterocycloalkyl, aryl, or aralkyl;

R₅ is H or alkyl; and

p is 1, 2, 3, 4, 5, 6, 7, or 8.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is —CH₂CH₂CN.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substitutedheterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is a nucleoside, nucleotide, oroligonucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is

wherein R′₁ represents independently for each occurrence alkyl, aryl,aralkyl, or —Si(R₄)₃; wherein said alkyl, aryl, and aralkyl group isoptionally substituted with —CN, —NO₂, —CF₃, or halogen; and n¹ is 1 to50 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 25 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 15 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 10 inclusive.

In certain embodiments, the present invention relates to theaforementioned method, wherein n¹ is 1 to 5 inclusive.

Agents for the Deprotection/Cleavage of Protecting Groups

RNA is often synthesized and purified by methodologies based on:tetrazole to activate the RNA amidite, NH₄OH to remove the exocyclicamino protecting groups, n-tetrabutylammonium fluoride (TBAF) to removethe 2′-OH alkylsilyl protecting groups, and gel purification andanalysis of the deprotected RNA. The RNA compounds may be formed eitherchemically or using enzymatic methods.

One important component of oligonucleotide synthesis is the installationand removal of protecting groups. Incomplete installation or removal ofa protecting group lowers the overall yield of the synthesis andintroduces impurities that are often very difficult to remove from thefinal product. In order to obtain a reasonable yield of a large RNAmolecule (i.e., about 20 to 40 nucleotide bases), the protection of theamino functions of the bases requires either amide or substituted amideprotecting groups. The amide or substituted amide protecting groups mustbe stable enough to survive the conditions of synthesis, and yetremovable at the end of the synthesis. These requirements are met by thefollowing amide protecting groups: benzoyl for adenosine, isobutyryl orbenzoyl for cytidine, and isobutyryl for guanosine. The amide protectinggroups are often removed at the end of the synthesis by incubating theRNA in NH₃/EtOH or 40% aqueous MeNH₂. In the case of the phenoxyacetyltype protecting groups on guanosine and adenosine and acetyl protectinggroups on cytidine, an incubation in ethanolic ammonia for 4 h at 65° C.is used to obtain complete removal of these protecting groups. However,deprotection procedures using mixtures of NH₃ or MeNH₂ are complicatedby the fact that both ammonia and methylamine are corrosive gases.Therefore, handling the reagents can be dangerous, particulary when thereaction is conducted at a large scale, e.g, manufacturing scale. Thevolatile nature of NH₃ and MeNH₂ also requires special procedures tocapture and neutralize any excess NH₃ and MeNH₂ once the deprotectionreaction is complete. Therefore, the need exists for less volatilereagents that are capable of effecting the amide deprotection reactionin high yield.

One aspect of the present invention relates to amino compounds withrelatively low volatility capable of effecting the amide deprotectionreaction. The classes of compounds with the aforementioned desirablecharacteristics are listed below. In certain instances, preferredembodiments within each class of compounds are listed as well.

1) Polyamines

The polyamine compound used in the invention relates to polymerscontaining at least two amine functional groups, wherein the aminefunctional group has at least one hydrogen atom. The polymer can have awide range of molecular weights. In certain embodiment, the polyaminecompound has a molecular weight of greater than about 5000 g/mol. Inother embodiments, the polyamine compound compound has a molecularweight of greater than about 10,000; 20,000, or 30,000 g/mol.

2) PEHA

3) PEG-NH₂

The PEG-NH₂ compound used in the invention relates to polyethyleneglycol polymers comprising amine functional groups, wherein the aminefunctional group has at least one hydrogen atom. The polymer can have awide range of molecular weights. In certain embodiment, the PEG-NH₂compound has a molecular weight of greater than about 5000 g/mol. Inother embodiments, the PEG-NH₂ compound has a molecular weight ofgreater than about 10,000; 20,000, or 30,000 g/mol.

4) Short PEG-NH₂

The short PEG-NH₂ compounds used in the invention relate to polyethyleneglycol polymers comprising amine functional groups, wherein the aminefunctional group has at least one hydrogen atom. The polymer has arelatively low molecular weight range.

5) Cycloalkylamines and Hydroxycycloalkyl Amines

The cycloalkylamines used in the invention relate to cycloalkylcompounds comprising at least one amine functional group, wherein theamine functional group has at least one hydrogen atom. Thehydroxycycloalkyl amines used in the invention relate to cycloalkylcompounds comprising at least one amine functional group and at leastone hydroxyl functional group, wherein the amine functional group has atleast one hydrogen atom. Representative examples are listed below.

6) Hydroxyamines

The hydroxyamines used in the invention relate to alkyl, aryl, andaralkyl compounds comprising at least one amine functional group and atleast one hydroxyl functional group, wherein the amine functional grouphas at least one hydrogen atom. Representative examples are9-aminononanol, 4-aminophenol, and 4-hydroxybenzylamine.

7) K₂CO₃/MeOH with or without Microwave

8) Cysteamine (H₂NCH₂CH₂SH) and Thiolated Amines 9)β-Amino-Ethyl-Sulfonic Acid, or the Sodium Sulfate ofβ-Amino-Ethyl-Sulfonic Acid

One aspect of the present invention relates to a method of removing anamide protecting group from an oligonucleotide, comprising the steps of:

admixing an oligonucleotide bearing an amide protecting group with apolyamine, PEHA, PEG-NH₂, Short PEG-NH₂, cycloalkyl amine,hydroxycycloalkyl amine, hydroxyamine, K₂CO₃/MeOH microwave,thioalkylamine, thiolated amine, β-amino-ethyl-sulfonic acid, or thesodium sulfate of β-amino-ethyl-sulfonic acid.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is an oligomer ofribonucleotides.

Reagents for Deprotection of a Silyl Group

As described in the previous section, the use of protecting groups is acritical component of oligonucleotide synthesis. Furthermore, theinstallation and removal of protecting groups must occur with high yieldto minimize the introduction of impurities into the final product. TheApplicants have found that the following reagents are superior forremoving a silyl protecting group during the synthesis of aoligonucleotide: pyridine-HF, DMAP-HF, urea-HF, ammonia-HF, ammoniumfluoride-HF, TSA-F, DAST, and polyvinyl pyridine-HF. For example, seeFIG. 7 and Example 5. Other aryl amine-HF reagents useful in thisinvention include compounds represented by AA:

wherein

R¹ is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R² is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and

R³ is aryl or heteroaryl.

For example, aryl amines of the hydrofluoride salts are selected fromthe group consisting of (dialkyl)arylamines, (alkyl)diarylamines,(alkyl)(aralkyl)arylamines, (diaralkyl)arylamines,(dialkyl)heteroarylamines, (alkyl)diheteroarylamines,(alkyl)(heteroaryl)arylamines, (alkyl)(heteroaralkyl)arylamines,(alkyl)(aralkyl)heteroarylamines, (diaralkyl)heteroarylamines,(diheteoroaralkyl)heteroarylamines, and(aralkyl)(heteroaralkyl)heteroarylamines.

In addition, the aforementioned methods can be practised with a hydrofluoride salt of a compound selected from the group consisting of

wherein, independently for each occurrence: X is O, S, NR¹ or CR₂; Y isN or CR; R is hydrogen, halogen, alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, aralkyl, heteroaralkyl, —C(═O)—, —C(═O)X—, —OR¹,—N(R¹)₂, —SR¹ or —(CH₂)_(m)—R¹; R¹ is hydrogen, halogen, alkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl orheteroaralkyl; and m is 0-10 inclusive.

In certain instances, the rate of the deprotection reaction can beexcelerated by conducting the deprotection reaction in the presence ofmicrowave radiation. As illustrated in Example 6, thetert-butyldimethylsilyl groups on a 10-mer or 12-mer could be removed in2 minutes or 4 minutes, respectively, by treatment with 1 M TBAF in THF,Et₃N—HF, or pyridine-HF/DBU in the presence of microwave radiation (300Watts, 2450 MHz).

One aspect of the present invention relates to a method removing a silylprotecting group from a oligonucleotide, comprising the steps of:

admixing an oligonucleotide bearing a silyl protecting group withpyridine-HF, DMAP-HF, Urea-HF, TSA-F, DAST, polyvinyl pyridine-HF, or anaryl amine-HF reagent of formula AA:

wherein

R¹ is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

R² is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and

R³ is aryl or heteroaryl.

In certain embodiments, the present invention relates to theaforementioned method, wherein said oligonucleotide is an oligomer ofribonucleotides.

In certain embodiments, the present invention relates to theaforementioned method, wherein the reaction is carried out in thepresence of microwave radiation.

Solid Supports for Oligonucleotide Synthesis

Solid-phase oligonucleotide synthesis is often performed on controlledpore glass. However, solid-phase oligonucleotide synthesis can becarried out on:

1) Fractosil

2) Non CPG, but silica based solid supports not including controlledpore glass

3) Universal linker on polystyrene beads.

4) Argogel

5) Argopore

6) AM Polystyrene

7) Novagel

8) PEGA; EM Merck poly(vinyl alcohol) (PVA); and Nitto Denko polystyrene

Experiments conducted using ArgoGel (dT succinate loaded on the support,loading=229.35 μmole/g) revealed that Poly-T synthesis was quite good.However, the material can be sticky leading to difficulties whenweighing and loading the column.

Experiments conducted using Argopore-1 (dT succinate loaded on thesupport, loading=322.14 μmole/g) revealed that the material exhibitedgood flow through, and the material was not sticky. However, thesynthesis coupling efficiency was reduced after 4-5 couplings.

Experiments conducted using Argopore-2 (dT succinate loaded on thesupport, loading=194 μmole/g) revealed that Poly-T synthesis was quitegood.

Linkers to Solid Supports

The oligonucleotide is generally attached to the solid support via alinking group. Suitable linking groups are an oxalyl linker, succinyl,dicarboxylic acid linkers, glycolyl linker, or thioglycolyl linker.Silyl linkers can also be used. See, e.g., DiBlasi, C. M.; Macks, D. E.;Tan, D. S. “An Acid-Stable tert-Butyldiarylsilyl (TBDAS) Linker forSolid-Phase Organic Synthesis” Org. Lett. 2005; ASAP Web Release Date:30 Mar. 2005; (Letter) DOI: 10.1021%1050370y. DiBlasi et al. describe arobust tert-butyldiarylsilyl (TBDAS) linker for solid-phase organicsynthesis. Importantly, the TBDAS linker is stable to aqueous HF inCH₃CN, which allows for the use of orthogonal HF-labile protectinggroups in solid-phase synthetic schemes. In one approach, theyestablished that cleavage of the linker could be achieved withtris(dimethylamino)-sulfonium (trimethylsilyl)-difluoride (TAS-F).

Solvents

In response to the growing emphasis on conducting reactions in solventsthat are more environmentally friendly, we have found thatoligonucleotides can be prepared using non-halogenated solvents. Forexample, oligonucleotides can be prepared using toluene,tetrahydrofuran, or 1,4-dioxane as the solvent.

RNA Synthesis Via H-Phosphonate Coupling

Synthesis of RNA using the H-phosphonate coupling method involvesreacting a nucleoside substituted with an H-phosphonate with thehydroxyl group of a second nucleoside in the presence of an activatingagent. One of the most commonly used activating agents is pivaloylchloride. However, pivaloyl chloride is not ideal for large-scalepreparations because it is flammable, corrosive, volatile (bp 105-106°C.), and has a relatively low flashpoint (Fp 8° C.). Therefore, the needexists for new activating agents devoid of the aforementioned drawbacks.

There are currently many useful condensing reagents known to the artskilled that are amenable to the H-phosphonate method of oligonucleotidesynthesis. See Wada et al. J. Am. Chem. Soc. 1997, 119, 12710-12721.Useful condensing reagents include acid chlorides, chlorophosphates,carbonates, carbonium type compounds and phosphonium type compounds. Ina preferred embodiment the condensing reagent is selected from a groupconsisting of pivaloyl chloride, adamantyl chloride,2,4,6-triisopropyl-benzenesulfonyl chloride,2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, diphenylphosphorochloridate, bis(2-oxo-3-oxazolidinyl)phosphinic chloride,bis(pentafluorophenyl)carbonate,2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, O-(azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,6-(trifluoromethyl)benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate and2-(benzotriazol-1-yloxy)-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphospholidiniumhexafluorophosphate. Additionally,2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinanane,NEP-Cl/pyridine/MeCN system has been described. See U.S. Pat. No.6,639,061.

The Applicants disclose herein other activating agents that can be usedin the H-phosphonate coupling method. Classes of compound that arebetter activating agents include acid chlorides of long-chain alkylgroups, acid chlorides of aromatic groups, acid chlorides of alkylgroups substituted with aromatic groups, and polymer bound acylchlorides. Representative examples of activiting agents are decanoylchloride, dodecanoyl chloride, benzoyl chloride, 1,2-dibenzyl ethanoylchloride, naphthoyl chloride, anthracenecarbonyl chloride, andfluorenecarbonyl chloride.

The Applicants disclose herein other oxidizing agents that can be usedin the H-phosphonate coupling method. One of the most common oxidizingagents is iodine. However, iodine is a very strong oxidizing agent thatcan lead to unwanted oxidation of sensitive functional groups on thenucleotide or oligonucleotide. Representative examples of oxidizingagents that can be used in the H-phosphonate coupling method include:camphorylsulfonyloxazaridine and N,O-bis(trimethylsilyl)-acetamide inMeCN/pyridine, CCl₄/pyridine/water/MeCN, and DMAP inpyridine/CCl₄/water.

Another aspect of the present invention relates to a method of forming aphosphodiester compound, comprising the steps of:

admixing a H-phosphonate, alcohol, and activating agent to form aphosphodiester compound, wherein said activating agent is selected fromthe group consisting of C₈-C₂₀ alkylcarbonyl chloride, arylcarbonylchloride, and aralkylcarbonyl chloride.

In certain embodiments, the present invention relates to theaforementioned method, wherein said activating agent is decanoylchloride, dodecanoyl chloride, benzoyl chloride, 1,2-dibenzyl ethanoylchloride, naphthoyl chloride, anthracenecarbonyl chloride, orfluorenecarbonyl chloride.

In certain embodiments, the present invention relates to theaforementioned method, wherein said H-phosphonate is represented byformula I:

wherein

R₁ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, or alkenyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substitutedheterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is a nucleoside or nucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is an optionally substitutedribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is a nucleoside, nucleotide,or oligonucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein said alcohol is represented by R₅—OH,wherein R₅ is optionally substituted alkyl, cycloalkyl,heterocycloalkyl, aryl, aralkyl, alkenyl, or—(C(R₆)₂)_(p)heterocycloalkyl; R₆ is H or alkyl; and p is 1, 2, 3, 4, 5,6, 7, or 8.

In certain embodiments, the present invention relates to theaforementioned method, wherein said phosphodiester is represented byformula J:

wherein

R₁ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, or alkenyl; and

R₂ is optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl,aralkyl, alkenyl, or —(C(R₆)₂)_(p)heterocycloalkyl; R₆ is H or alkyl;and p is 1, 2, 3, 4, 5, 6, 7, or 8.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substitutedheterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₁ is a nucleoside or nucleotide.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is (C(R₆)₂)_(p)heterocycloalkyl.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituted ribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is an optionally substituteddeoxyribose.

In certain embodiments, the present invention relates to theaforementioned method, wherein R₂ is a nucleoside or nucleotide.

Purification of Double-Stranded RNA

One common problem encountered in RNA preparation is obtaining thedesired oligonucleotide in high purity. In many cases, reactions used toprepare the oligonucleotide do not achieve 100% conversion, or theygenerate side-products. Unfortunately, the unreacted starting materialsand side-products often have similar chemical properties, making it verydifficult to separate the desired product from these impurities.

The most quantitative procedure for recovering a fully deprotected RNAmolecule is by either ethanol precipitation, or an anion exchangecartridge desalting, as described in Scaringe et al. Nucleic Acids Res.1990, 18, 5433-5341. Purification of long RNA sequences is oftenperformed using a two-step chromatographic procedure in which themolecule is first purified on a reverse phase column with either thetrityl group at the 5′ position on or off. This purification is carriedout using an acetonitrile gradient with triethylammonium or bicarbonatesalts as the aqueous phase. In the case where the trityl group is stillattached to the RNA during purification, the trityl group may be removedby the addition of an acid and drying of the partially purified RNAmolecule. The final purification is carried out on an anion exchangecolumn, using alkali metal perchlorate salt gradients to elute the fullypurified RNA molecule as the appropriate metal salts, e.g. Na⁺, Li⁺ etc.A final de-salting step on a small reverse-phase cartridge completes thepurification procedure.

In certain instances, purification of long RNA molecules is carried outusing anion exchange chromatography, particularly in conjunction withalkali perchlorate salts. This system is used to purify very long RNAmolecules. In particular, it is advantageous to use a Dionex NUCLEOPAK100® or a Pharmacia MONO Q® anion exchange column for the purificationof RNA by the anion exchange method. This anion exchange purificationmay be used following a reverse-phase purification or prior toreverse-hase purification. This method results in the formation of asodium salt of the ribozyme during the chromatography. Replacement ofthe sodium alkali earth salt by other metal salts, e.g., lithium,magnesium or calcium perchlorate, yields the corresponding salt of theRNA molecule during the purification.

In the case of the two-step purification procedure wherein the firststep is a reverse-phase purification followed by an anion exchange step,the reverse-phase purification is usually perfomed using polymeric,e.g., polystyrene based, reverse-phase media using either a 5′-trityl-onor 5′-trityl-off method. Either molecule may be recovered using thisreverse-phase method, and then, once detritylated, the two fractions maybe pooled and submitted to an anion exchange purification step asdescribed above.

However, many synthetic RNA products still contain substantialquantities of impurities despite performing the arduous purificationsteps, as described above. Therefore, the need exists for a newpurification procedure to provide RNA in a highly pure form.

The Applicants have surprising discovered that impurities in acomposition of single-stranded RNA can be readily removed by HPLCpurification of a mixture of single-stranded RNA that has been annealedto generate double-stranded RNA. A diagram illustrating the overallprocedure is presented in FIG. 9. The structure of AL-4112, AL-4180,AL-DP-4014, AL-2200, AL-22-1, AL-DP-4127, AL-2299, AL-2300, AL-DP-4139,AL-2281, AL-2282, and AL-DP-4140 is presented in FIG. 10. The specificprocedure for the purification of AL-DP-4014, the components of whichare AL-4112 and AL-4180, is shown in FIGS. 11 and 12. AL-DP-4127,AL-DP-4139, and AL-DP-4140 were also purified using the proceduresdescribed in FIGS. 9, 11, and 12. The results from the analyses arepresented in FIGS. 19-39.

Alternative procedures of RNA purification using the two-strand methodare presented in FIGS. 40-43.

One aspect of the present invention relates to a method of purifying anoligonucleotide, comprising the steps of:

annealing a first oligonucleotide with a second oligonucleotide to forma double-stranded oligonucleotide, subjecting said double-strandedoligonucleotide to chromatographic purification.

In certain embodiments, the present invention relates to theaforementioned method, wherein said annealing a first oligonucleotidewith a second oligonucleotide is done at a temperature between a firsttemperature and a second temperature, wherein said first temperature isabout the T_(m) of a double-stranded oligonucleotide consisting of saidfirst oligonucleotide and a third oligonucleotide, wherein said thirdoligonucleotide is the antisense sequence corresponding to the firstoligonucleotide, and said second temperature is about 5 degrees belowsaid first temperature.

In certain embodiments, the present invention relates to theaforementioned method, wherein said chromatographic purification isliquid chromatography.

In certain embodiments, the present invention relates to theaforementioned method, wherein said chromatographic purification ishigh-performance liquid chromatography.

In certain embodiments, the present invention relates to theaforementioned method, wherein said first oligonucleotide is an oligomerof ribonucleotides.

In certain embodiments, the present invention relates to theaforementioned method, wherein said second oligonucleotide is anoligomer of ribonucleotides.

In certain embodiments, the present invention relates to theaforementioned method, wherein said first oligonucleotide is an oligomerof ribonucleotides, and said second oligonucleotide is an oligomer ofribonucleotides.

RNA HPLC Methods

As described above, high-peformance liquid chromatography (HPLC) is animportant method used in the purification of RNA compounds. A largevariety of columns, solvents, additives, and conditions have beenreported for purifying oligonucleotides. However, current procedures forpurifying RNA compounds are not able to separate the RNA compound fromsignificant amounts of impurities. The Applicants report hereinimprovements to existing HPLC procedures thereby providing the RNAcompound with substantially fewer impurities:

1) Use tetrabutylammonium acetate as ion-pairing agent in analyticalHPLC separations of oligonucleotides. See M. Gilar for use oftetrabutylammonium acetate in analytical HPLC separations. M. GilarAnalytical Biochemistry 2001, 298, 196-206.

2) HPLC purification in DMT-off mode with C-18 column or C-4 column forlipophilic conjugates of RNA compounds.

3) HPLC purification of RNA compounds using ethanol or acetonitrile asthe solvent.

2′-Protecting Groups for RNA Synthesis

As described above, protecting groups play a critical role in RNAsynthesis. The Applicants describe herein several new protecting groupsthat can be used in RNA synthesis. One class of 2′-protecting groupsthat can be used in RNA synthesis is carbonates. One preferred carbonateis propargyl carbonate shown below.

The propargyl carbonate can be removed using benzyltriethylammoniumtetrathiomolybdate as described in Org. Lett. 2002, 4, 4731.

Another class of 2′-protecting groups that can be used in RNA synthesisis acetals. Acetal groups can be deprotected using aqueous acid. Severalrepresentative acetal protecting groups are shown below. See FIG. 44 foradditional examples.

Other 2′-protecting groups that can be used in RNA synthesis are shownbelow.

In addition, a bis-silyl strategy could be used in RNA synthesis. Thisstrategy involves protecting both the 2′-hydroxyl group of the riboseand the phosphate attached to the 3′-position of the ribose with a silylgroup. A representative example is presented below in FIG. 44.

Representative examples of the above-mentioned protecting groups onvarious nucleosides are presented in FIG. 44.

Alternate 5′-Protecting Groups

In place of dimethoxytrityl (DMT), monomethoxytrityl (MMT),9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox)and their analogs can be employed.

Alternate Base-Protecting Groups

1) Nps and DNPS groups (Fukuyama)

2) phenacetyl (removal by penicillin G acylase)

Enzymatic Methods for Removal of Protecting Groups

Another aspect of the present invention relates to protecting groupswhich can be removed enzymatically. Aralkyl esters represented by—O₂CCH₂R, wherein R is phenyl, pyridinyl, aniline, quinoline, orisoquinoline can be removed from the 2′-position of a nucleoside byenzymatic cleavage using penicillin G acylase. Representative examplesof nucleosides bearing aralkyl ester protecting groups at the2′-position of the ribose ring are presented in FIG. 45. In addition,certain internal amidites, including those shown in FIG. 45, can beremoved by enzymatic cleavage.

One aspect of the present invention relates to a method of removing aprotecting group, comprising the steps of:

admixing an optionally substituted ribose bearing a protecting group atthe C2 position with an enzyme to produce an optionally substitutedribose bearing a hydroxyl group at the C2 position.

In certain embodiments, the present invention relates to theaforementioned method, wherein said protecting group is an aralkylester.

In certain embodiments, the present invention relates to theaforementioned method, wherein said protecting group is represented bythe formula —O₂CCH₂R, wherein R is phenyl, pyridinyl, aniline,quinoline, or isoquinoline.

In certain embodiments, the present invention relates to theaforementioned method, wherein said enzyme is penicillin G acylase.

In certain embodiments, the present invention relates to theaforementioned method, wherein said ribose is a ribonucleotide oligomer.

Synthesis of Oligonucleotides Containing a TT Unit

In certain embodiments, it is preferable to prepare an oligonucleotidecomprising two adjacent thymidine nucleotides. In a more preferredembodiment, the thymidine nucleotides are located at the 3′ end of theoligonucleotide. The thymidine-thymidine (TT) nucleotide unit can beprepared using solution-phase chemistry, and then the TT unit isattached to a solid support. In certain embodiments, the TT unit islinked via a phosphorothioate group. In certain instances, the differentstereoisomers of the phosphorothioate TT unit may be separated prior toattachment of the TT unit to the solid support. The remainder of theoligonucleotide strand can be synthesized via standard solid-phasesynthesis techniques using the TT-support bound unit as a primer. Incertain instances, the thymidine-thymidine nucleotide unit is made ofdeoxythymidine residues.

One aspect of the present invention relates to a method of preparing anoligonucleotide comprising a dinucleoside unit, comprising the steps of:

synthesizing a dinucleoside group via solution-phase chemistry,attaching said dinucleoside group to a solid support to form a primer,adding additional nucleotides to said primer using solid-phase synthesistechniques.

In certain embodiments, the present invention relates to theaforementioned method, wherein each nucleoside residue of saiddinucleoside group is independently a natural or unnatural nucleoside.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises twonucleoside residues each independently comprising a sugar and anucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and saidnucleobase is natural or unnatural.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises twonucleoside residues each independently comprising a sugar and anucleobase, wherein said sugar is an L-ribose or L-deoxyribose, and saidnucleobase is natural or unnatural.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises twothymidine residues.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises twodeoxythymidine residues.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises two2′-modified 5-methyl uridine or uridine residues, wherein the2′-modifications are 2′-O-TBDMS, 2′-OMe, 2′-F, 2′-O—CH2-CH2-O-Me, or2′-O-alkylamino derivatives.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises aphosphorothioate linkage, phosphorodithioate linkage, alkyl phosphonatelinkage, or boranophosphate linkage.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises aphosphorothioate linkage, alkyl phosphonate linkage, or boranophosphatelinkage; and said dinucleoside group is a single stereoisomer at thephosphorus atom.

In certain embodiments, the present invention relates to theaforementioned method, wherein the linkage between the nucleosideresidues of said dinucleoside group is a 3′-5′ linkage.

In certain embodiments, the present invention relates to theaforementioned method, wherein the linkage between the nucleosideresidues of said dinucleoside group is a 2′-5′ linkage.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises twonucleoside residues each independently comprising a sugar and anucleobase, wherein said sugar is a D-ribose or D-deoxyribose, and saidnucleobase is natural or unnatural; and the linkage between thenucleoside residues of said dinucleotide group is unnatural andnon-phosphate.

In certain embodiments, the present invention relates to theaforementioned method, wherein said dinucleoside group comprises twonucleoside residues each independently comprising a sugar and anucleobase, wherein said sugar is an L-ribose or L-deoxyribose, and saidnucleobase is natural or unnatural; and the linkage between thenucleoside residues of said dinucleotide group is MMI, amide linkage, orguanidinium linkage.

Improved Procedures for the Synthesis of Nucleosides, Nucleotides, andOligonucleotides

Importantly, any one of the above-mentioned improvements can be usedalone with standard methods of preparing nucleosides, nucleotides, andoligonucleotides, or more than one of the above-mentioned improvementscan be used together with standard methods of preparing nucleosides,nucleotides, and oligonucleotides. Furthermore, one of ordinary skill inthe art can readily determine the optimal conditions for each of theimprovements described above.

General Description of Oligonucleotides

As described above, the present invention relates to processes andreagents for oligonucleotide synthesis and purification. The followingdescription is meant to briefly describe some of the major types andstructural features of oligonucleotides. Importantly, the followingsection is only representative and not meant to limit the scope of thepresent invention.

Oligonucleotides can be made of ribonucleotides, deoxyribonucleotides,or mixtures of ribonucleotides and deoxyribonucleotides. The nucleotidescan be natural or unnatural. Oligonucleotides can be single stranded ordouble stranded. Various modifications to the sugar, base, and phosphatecomponents of oligonucleotides are described below. As defined here,oligonucleotides having modified backbones or internucleoside linkagesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone. For the purposes theinvention, modified oligonucleotides that do not have a phosphorus atomin their intersugar backbone can also be considered to beoligonucleosides.

Specific oligonucleotide chemical modifications are described below. Itis not necessary for all positions in a given compound to be uniformlymodified, and in fact more than one of the following modifications maybe incorporated in a single siRNA compound or even in a singlenucleotide thereof.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalklyphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents that teach the preparation of theabove phosphorus atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleoside units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigonucleotide, an oligonucleotide mimetic, that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

Some preferred embodiments of the present invention employoligonucleotides with phosphorothioate linkages and oligonucleosideswith heteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and theamide backbones of the above referenced U.S. Pat. No. 5,602,240. Alsopreferred are oligonucleotides having morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Oligonucleotides may additionally or alternatively comprise nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al. Angewandte Chemie,International Edition 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above-noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are herebyincorporated by reference.

The oligonucleotides may additionally or alternatively comprise one ormore substituted sugar moieties. Preferred oligonucleotides comprise oneof the following at the 2′ position: OH; F; O-, S-, or N-alkyl, O-, S-,or N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al. Helv. Chim.Acta 1995, 78, 486), i.e., an alkoxyalkoxy group. a further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533,filed on Jan. 30, 1998, the contents of which are incorporated byreference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-O-methoxyethyl, 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides.

As used herein, the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′-position of the ribofuranosylmoiety with or without an oxygen atom. Sugar substituent groups include,but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole andpolyethers of the formula (O-alkyl)_(m), wherein m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi et al. (Drug Design and Discovery1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardoet. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992,9:249), each of which is hereby incorporated by reference in itsentirety. Further sugar modifications are disclosed by Cook (Anti-CancerDrug Design, 1991, 6, 585-607). Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl, and alkyl amino substitution is describedin U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” herebyincorporated by reference in its entirety.

Additional sugar substituent groups amenable to the present inventioninclude 2′-SR and 2′-NR₂ groups, wherein each R is, independently,hydrogen, a protecting group or substituted or unsubstituted alkyl,alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No.5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in itsentirety. The incorporation of 2′-SR monomer synthons is disclosed byHamm et al. (J. Org. Chem. 1997, 62, 3415-3420). 2′-NR nucleosides aredisclosed by Goettingen, M. J. Org. Chem., 1996, 61, 6273-6281; andPolushin et al. Tetrahedron Lett. 1996, 37, 3227-3230. Furtherrepresentative 2′-substituent groups amenable to the present inventioninclude those having one of formula I or II:

wherein,

E is C₁-C₁₀ alkyl, N(Q₃)(Q₄) or N═C (Q₃)(Q₄); each Q₃ and Q₄ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport; or Q₃ and Q₄, together, form a nitrogen protecting group or aring structure optionally including at least one additional heteroatomselected from N and O;

q₁ is an integer from 1 to 10;

q₂ is an integer from 1 to 10;

q₃ is 0 or 1;

q₄ is 0, 1 or 2;

each Z₁, Z₂ and Z₃ is, independently, C₄-C₇ cycloalkyl, C₅-C₁₄ aryl orC₃-C₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl groupis selected from oxygen, nitrogen and sulfur;

Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is, independently, H, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H orC₁-C₈ alkyl; and

Z₅ is C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆-C₁₄ aryl, N(Q₃)(Q₄), OQ₃, halo, SQ₃ or CN.

Representative 2′-O-sugar substituent groups of formula I are disclosedin U.S. Pat. No. 6,172,209, entitled “Capped 2′-OxyethoxyOligonucleotides,” hereby incorporated by reference in its entirety.Representative cyclic 2′-O-sugar substituent groups of formula II aredisclosed in U.S. Pat. No. 6,271,358, filed Jul. 27, 1998, entitled “RNATargeted 2′-Modified Oligonucleotides that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O include,but are not limited to, NH, NR, S, CH₂, CHF, and CF₂. See, e.g., Secristet al., Abstract 21, Program & Abstracts, Tenth InternationalRoundtable, Nucleosides, Nucleotides and their Biological Applications,Park City, Utah, Sep. 16-20, 1992.

Oligonucleotides may also have sugar mimetics, such as cyclobutylmoieties, hexoses, cyclohexenyl in place of the pentofuranosyl sugar.Representative United States patents that teach the preparation of suchmodified sugars structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,700,920; and 5,859,221, all of which are herebyincorporated by reference.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide. For example, one modification of oligonucleotidesinvolves chemically linking to the oligonucleotide one or moreadditional moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties, such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053),a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of whichis herein incorporated by reference.

Oligonucleotides can be substantially chirally pure with regard toparticular positions within the oligonucleotides. Examples ofsubstantially chirally pure oligonucleotides include, but are notlimited to, those having phosphorothioate linkages that are at least 75%Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidateor phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and5,521,302).

Synthetic RNA molecules and derivatives thereof that catalyze highlyspecific endoribonuclease activities are known as ribozymes. (See,generally, U.S. Pat. No. 5,543,508 to Haseloff et al., issued Aug. 6,1996, and U.S. Pat. No. 5,545,729 to Goodchild et al., issued Aug. 13,1996.) The cleavage reactions are catalyzed by the RNA moleculesthemselves. In naturally occurring RNA molecules, the sites ofself-catalyzed cleavage are located within highly conserved regions ofRNA secondary structure (Buzayan et al., Proc. Natl. Acad. Sci. U.S.A.,1986, 83, 8859; Forster et al., Cell, 1987, 50, 9). Naturally occurringautocatalytic RNA molecules have been modified to generate ribozymeswhich can be targeted to a particular cellular or pathogenic RNAmolecule with a high degree of specificity. Thus, ribozymes serve thesame general purpose as antisense oligonucleotides (i.e., modulation ofexpression of a specific gene) and, like oligonucleotides, are nucleicacids possessing significant portions of single-strandedness. That is,ribozymes have substantial chemical and functional identity witholigonucleotides and are thus considered to be equivalents for purposesof the present invention.

In certain instances, the oligonucleotide may be modified by a moiety. Anumber of moieties have been conjugated to oligonucleotides in order toenhance the activity, cellular distribution or cellular uptake of theoligonucleotide, and procedures for performing such conjugations areavailable in the scientific literature. Such moieties have includedlipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad.Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med.Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such oligonucleotideconjugates have been listed above. Typical conjugation protocols involvethe synthesis of oligonucleotides bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with theoligonucleotide still bound to the solid support or following cleavageof the oligonucleotide in solution phase. Purification of theoligonucleotide conjugate by HPLC typically affords the pure conjugate.

One type of double-stranded RNA is short interfering RNA (siRNA). Incertain embodiments, the backbone of the oligonucleotide can be modifiedto improve the therapeutic or diagnostic properties of the siRNAcompound. The two strands of the siRNA compound can be complementary,partially complementary, or chimeric oligonucleotides. In certainembodiments, at least one of the bases or at least one of the sugars ofthe oligonucleotide has been modified to improve the therapeutic ordiagnostic properties of the siRNA compound.

The siRNA agent can include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the siRNA agent, or a fragment thereof, can mediate down regulationof the target gene. It will be understood that the term “ribonucleotide”or “nucleotide” can, in the case of a modified RNA or nucleotidesurrogate, also refer to a modified nucleotide, or surrogate replacementmoiety at one or more positions. Thus, the siRNA agent is or includes aregion which is at least partially complementary to the target RNA. Incertain embodiments, the siRNA agent is fully complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the siRNA agent and the target, but the correspondence must besufficient to enable the siRNA agent, or a cleavage product thereof, todirect sequence specific silencing, such as by RNAi cleavage of thetarget RNA. Complementarity, or degree of homology with the targetstrand, is most critical in the antisense strand. While perfectcomplementarity, particularly in the antisense strand, is often desiredsome embodiments can include one or more but preferably 6, 5, 4, 3, 2,or fewer mismatches with respect to the target RNA. The mismatches aremost tolerated in the terminal regions, and if present are preferably ina terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the over alldouble-strand character of the molecule.

In addition, an siRNA agent will often be modified or include nucleosidesurrogates. Single stranded regions of an siRNA agent will often bemodified or include nucleoside surrogates, e.g., the unpaired region orregions of a hairpin structure, e.g., a region which links twocomplementary regions, can have modifications or nucleoside surrogates.Modification to stabilize one or more 3′- or 5′-terminus of an iRNAagent, e.g., against exonucleases, or to favor the antisense sRNA agentto enter into RISC are also favored. Modifications can include C3 (orC6, C7, C12) amino linkers, thiol linkers, carboxyl linkers,non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol,hexaethylene glycol), special biotin or fluorescein reagents that comeas phosphoramidites and that have another DMT-protected hydroxyl group,allowing multiple couplings during RNA synthesis.

siRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed sRNA agents or shorter iRNA agentsherein. “sRNA agent or shorter iRNA agent” as used refers to an iRNAagent that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The sRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

Each strand of a sRNA agent can be equal to or less than 30, 25, 24, 23,22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length. The strand ispreferably at least 19 nucleotides in length. For example, each strandcan be between 21 and 25 nucleotides in length. Preferred sRNA agentshave a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotidepairs, and one or more overhangs, preferably one or two 3′ overhangs, of2-3 nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an siRNA agent will preferably have one or more of thefollowing properties:

(1) it will, despite modifications, even to a very large number, or allof the nucleosides, have an antisense strand that can present bases (ormodified bases) in the proper three dimensional framework so as to beable to form correct base pairing and form a duplex structure with ahomologous target RNA which is sufficient to allow down regulation ofthe target, e.g., by cleavage of the target RNA;

(2) it will, despite modifications, even to a very large number, or allof the nucleosides, still have “RNA-like” properties, i.e., it willpossess the overall structural, chemical and physical properties of anRNA molecule, even though not exclusively, or even partly, ofribonucleotide-based content. For example, an siRNA agent can contain,e.g., a sense and/or an antisense strand in which all of the nucleotidesugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, theelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C_(3′)-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix. Further, since fluorine is agood hydrogen bond acceptor, it can participate in the same hydrogenbonding interactions with water molecules that are known to stabilizeRNA structures. Generally, it is preferred that a modified moiety at the2′ sugar position will be able to enter into H-bonding which is morecharacteristic of the OH moiety of a ribonucleotide than the H moiety ofa deoxyribonucleotide. A preferred siRNA agent will: exhibit aC_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of itssugars; exhibit a C_(3′)-endo pucker in a sufficient amount of itssugars that it can give rise to a the RNA-characteristic A-family-typehelix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which isnot a C_(3′)-endo pucker structure.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. A single strand iRNA agent should besufficiently long that it can enter the RISC and participate in RISCmediated cleavage of a target mRNA. A single strand iRNA agent is atleast 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50nucleotides in length. It is preferably less than 200, 100, or 60nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill preferably be equal to or less than 200, 100, or 50, in length.Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin will preferablyhave a single strand overhang or terminal unpaired region, preferablythe 3′, and preferably of the antisense side of the hairpin. Preferredoverhangs are 2-3 nucleotides in length.

Chimeric oligonucleotides, or “chimeras,” are oligonucleotides whichcontain two or more chemically distinct regions, each made up of atleast one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. Consequently, comparable results can often beobtained with shorter oligonucleotides when chimeric oligonucleotidesare used, compared to phosphorothioate oligodeoxynucleotides. Chimericoligonucleotides of the invention may be formed as composite structuresof two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such oligonucleotides have also been referred to in the art as hybridsor gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356;5,700,922; and 5,955,589, each of which is herein incorporated byreference. In certain embodiments, the chimeric oligonucleotide isRNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, or RNA-DNA-RNA-DNA, whereinthe oligonucleotide is between 5 and 60 nucleotides in length.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group). Forexample, a benzyl group (PhCH₂—) is an aralkyl group.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, anthracene, naphthalene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring can be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thio lane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a group permittedby the rules of valence.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonylamino” is art recognized and includes a moiety thatcan be represented by the general formula:

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

The term “sulfonyl”, as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term “sulfoxido” as used herein, refers to a moiety that can berepresented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of -Se-alkyl,-Se-alkenyl, -Se-alkynyl, and -Se-(CH₂)_(m)—R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. In addition, the substituentcan be halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, and the like. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. This invention is not intendedto be limited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts,P.G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: NewYork, 1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., functioning as analgesics), whereinone or more simple variations of substituents are made which do notadversely affect the efficacy of the compound in binding to sigmareceptors. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants which are in themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Oligonucleotide Synthesis Using Phosphoramidite Activators35-48 (see FIGS. 1-3)

In certain instances the strength of the activator is increased byforming the activated salt resulting in decreased coupling time for RNASynthesis.

A decamer RNA molecules (49, 5′-CAUCGCTGAdT-3′ SEQ ID NO: 7) wassynthesized on a 394 ABI machine (ALN 0208) using the standard 98 stepcycle written by the manufacturer with modifications to a few wait stepsas described below. The solid support was controlled pore glass (CPG,prepacked, 1 μmole, 500 {acute over (Å)}, Proligo Biochemie GmbH) andthe monomers were RNA phosphoramidites with fast deprotecting groupsobtained from Pierce Nucleic Acid Technologies used at concentrations of0.15 M in acetonitrile (CH₃CN) unless otherwise stated. Specifically theRNA phosphoramidites were5′-O-Dimethoxytrityl-N⁶-phenoxyacetyl-2′-O-tbutyldimethylsilyl-adenosine-3′-O-(B-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N²-p-isopropylphenoxyacetyl-2′-O-tbutyldimethylsilyl-guanosine-3′-O-(B-cyanoethyl-N,N′-diisopropyl)phosphoramidite,5′-O-Dimethoxytrityl-N⁴-acetyl-2′-O-tbutyldimethylsilyl-cytidine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)phosphoramidite,and5′-O-Dimethoxytrityl-2′-O-tbutyldimethylsilyl-uridine-3′-O-(β-cyanoethyl-N,N′-diisopropyl)-phosphoramidite;

The coupling times were either 1, 3 or 5 minutes for the different saltconcentrations which themselves were 10, 20 and 40 mol % relative to the5-(ethylthio)-1H-tetrazole (ETT, 0.25 M, Glen Research).Diisopropylammonium salt of ETT with required mol % was obtained byadding calculated amount of anhydrous diisopropylamine to 0.25 M ETTsolution and stored over molecular sieves for 4-6 h. Details of theother reagents are as follows: Cap A: 5% Phenoxyaceticanhydride/THF/pyridine, (Glen Research, & Cap B: 10%N-methylimidazole/THF, (Glen Research); Oxidant 0.02 M Iodine inTHF/Water/Pyridine (Glen Research] Detritylation was achieved with 3%TCA/dichloromethane (Proligo).

After completion of synthesis the CPG was transferred to a screw capRNase free microfuge tube. The oligonucleotide was cleaved from the CPGwith simultaneous deprotection of base and phosphate groups with 1.0 mLof a mixture of 40% methylamine:ammonia (1:1)] for 30 minutes at 65° C.The solution was then lyophilized to dryness.

Example 2 Synthesis of Compound 1 (R′=H and R″=C(S)OEt or R′,R″=H)

A solution of chlorocarbonyl sulfenyl chloride (8.4 mL, 0.1 mol) in dryether (50 mL) was added dropwise to a cold solution of thiourea (7.62 g,0.1 mol) in dry ether (500 mL) and triethylamine (14 mL, 0.1 mol) cooledwith ice-bath in 3 h under an argon atmosphere. The reaction mixture wasstirred at the same temperature for total of 6 h. The solids werefiltered off and the filtration was concentrated into a crude residuewhich was further crystallized with dichlorometrhane-hexanes to give apure compound (2.5 g). The mother liquid was then concentrated into acrude residue which was applied to a column of silica gel eluted withdichloromethane-methanol (40:1) to give a pure compound (180 mg). Thetotal yield is about 30%. ¹H-NMR (CDCl₃, 400 MHz): δ 10.46 (br, 1H),4.38 (q, 2H, J=6.8, 14.4 Hz, CH₂), 1.39 (t, 3H, J=7.2 Hz, CH₃). ¹³C-NMR(CDCl₃, 100 MHz): 181.01, 177.00, 153.75, 64.68, 14.32.

Example 3 Phosphorothioation of di- and Poly-Oligothymidine Using SulfurTransfer Reagent 1 (R′=H and R″=C(S)OEt or R′,R″=H)

Dinucleotide 2 and hexamer 3 were synthesized on a 394 ABI machine usingthe standard 93 step cycle written by the manufacturer withmodifications to a few wait steps as described below. Activator used was5-(ethylthio)-1H-tetrazole (0.25 M), and for PS-oxidation, 0.05 M 1 inanhydrous acetonitrile was used. The sulfurization time was about 4 min.After completion of the synthesis, 2 and 3 were deprotected from supportby aqueous ammonia treatment at 55° C. for 1 h. After HPLC purification,the compound were analysed by LC-MS.

The results of phosphorothioation of oligothymdine using 1 as thesulfur-transfer agent are shown below.

Mass Mass Compound Sequence, all P═S Calc. Found 2 5′ TT 3′ 562.46562.22 3 5′ TTTTTT 3′ 1843.52 1842.05

Example 4 Medium/Large Scale Oligonucleotides Synthesis with P═O, P═Sand P═O/P═S Mixed Backbone A. Solid Phase Synthesis of Sequences 23 withP═O Backbone and 24 with P═S Backbone

200 μmole syntheses were performed on the ÅKTA OligoPilot 100 in 6.3 mLcolumns using 500 Å dT-CPG loaded at 97 μmole/g (Prime Synthesis; Aston,Pa.) Detritylation was performed with 3% dichloroacetic acid (DCA) indichloromethane (CH₂Cl₂.) Coupling was accomplished with 2 eq. of DNA3′-β-cyanoethylphosphoramidites (CEP) or 2.5 eq. RNA3′-β-cyanoethylphosphoramidites (Pierce Nucleic Acids; Milwaukee, Wis.)used at 0.2 M in acetonitrile (MeCN). Activator was 0.6 M5-Ethylthiotetrazole (American International Chemical; Natick, Mass.) inMeCN and was used at three-fold excess relative to RNA CEPs and at4.5-fold excess to DNA CEPs. Oxidation was via 50 mM I₂ in 90% pyridine10% H₂O or with 0.05 M 3-ethoxy-1,2,4-dithiazolidine-5-one (EDITH) inMeCN (Q. Xu, et al. Nucleic Acids Research, Vol. 24, No. 18, pp.3643-3644). Capping was with 10% acetic anhydride (Ac₂O) 10%1-methylimidazole (1-MeIm) 15% 2,6-lutidine in MeCN.

After synthesis, support was deblocked in 25 mL 40% methylamine (MeNH₂)in H₂O for 20 minutes at 60° C. and 200 rpm, then chilled in dry ice[CO₂(s)] and the support filtered off in a sintered glass funnel andrinsed with 75 mL dimethylsulfoxide (DMSO) added to the filtrate. Tothis solution was added 25 mL triethylammonium trihydrofluoride(TEA.3HF, TREAT) followed by heating to 60° C. for 20 minutes at 200rpm. After chilling in CO₂(s) this solution was diluted with 125 mL 20mM sodium acetate (NaOAc) and pH 6 confirmed. If necessary, pH wasadjusted with HCl.

Analysis was performed on an Agilent 1100 series HPLC using a Dionex4×250 mm DNAPak column. Buffer A was 1 mM EDTA, 25 mM Tris pH 8, 20 mMNaClO₄. Buffer B was 1 mM EDTA, 25 mM Tris pH 8, 0.4 M NaClO₄.Separation was performed on a 0-40% B gradient with buffers and columnheated to 65° C.

Materials were purified on an ÅKTA Explorer equipped with a XK26/10column (Amersham Biosciences; Piscataway, N.J.) packed to a bed heightof 10 cm with Hi Load Q Sepharose. Buffer A was 1 mM EDTA, 25 mM Tris pH8. Buffer B was 1 mM EDTA, 25 mM Tris pH8, 0.4 M NaClO₄. Crude materialswere diluted 4-6 fold with H₂O and loaded. Pooled purified material=8.1kAU at 96% by ion exchange (IEX).

The solutions containing the crude material were diluted 4-6 fold,loaded onto the column in 1-3 kAU amounts at 10 mL/min and eluted with asegmented gradient from 0-60% B. Appropriate fractions were pooled andthis pooled material desalted in 30 mL amounts over Sephadex G-25 on aBioPilot column (6 cm dia.×7.5 cm) against H₂O. The eluate was vacuumevaporated to less than 25 mL, shell frozen and lyophilized.

The results from the synthesis of 23 and 24 are presented below. Notethat purification was performed on an ÅKTA Explorer and that “nd”indicates that the value was not determined.

Thiolation crude Sequence Agent Quantity % Y % f1 IEX Purification 23 —— 43 nd 8.6 kAU @ 94% fl 24 0.05 M 1CV in 42 71 8.1 kAU (=48% EDITH1 min. of crude) @ 96% fl 23 = 5′-GCGGAUCAAACCUCACCAAdTdT-3′ (SEQ ID NO:3) 24 = 5′-UUGGUGAGGUUUGAUCCGCdTdT-3′ (SEQ ID NO: 4)

B. Solid phase synthesis of mixed phosphorothioate-phosphodiesteroligoribonucleotides using phenyl acetyl disulfide or3-ethoxy-1,2,4-dithiazo line-5-one

200 μmole syntheses were performed on the ÅKTA OligoPilot 100 in 6.3 mLcolumns using 500 Å dT-CPG loaded at 97 μmole/g (Prime Synthesis; Aston,Pa.) Detritylation was performed with 3% dichloroacetic acid (DCA) indichloromethane (CH₂Cl₂.) Coupling was accomplished with 2 eq. of DNACEPs or 2.5 eq. of RNA CEPs (Pierce Nucleic Acids; Milwaukee, Wis.) usedat 0.2 M in acetonitrile (MeCN.) Activator was 0.6 M5-Ethylthiotetrazole (American International Chemical; Natick, Mass.) inMeCN and was used at threefold excess relative to RNA CEPs and at4.5-fold excess to DNA CEPs. Oxidation was via 50 mM I₂ in 90% pyridine10% H₂O. Thiolation was with 0.2 M phenyl acetyl disulfide (PADS) in 1:13-picoline:MeCN or with 0.05 M 3-ethoxy-1,2,4-dithiazolidine-5-one(EDITH) in MeCN (Q. Xu, et al. Nucleic Acids Research, Vol. 24, No. 18,pp. 3643-3644.) Capping was with 10% acetic anhydride (Ac₂O) 10%1-methylimidazole (1-MeIm) 15% 2,6-lutidine in MeCN. When EDITH wasused, capping was performed both before and after the thiolationreaction (M. Ma, et al. Nucleic Acids Research, 1997, Vol. 25, No. 18,pp. 3590-3593).

After synthesis, support was deblocked in 25 mL 40% methylamine (MeNH₂)in H₂O for 20 minutes at 60° C. and 200 rpm, then chilled in dry ice[CO₂(s)] and the support filtered off in a sintered glass funnel andrinsed with 75 mL dimethylsulfoxide (DMSO) added to the filtrate. Tothis solution was added 25 mL triethylammonium trihydrofluoride(TEA.3HF, TREAT) followed by heating to 60° C. for 20 minutes at 200rpm. After chilling in CO₂(s) this solution was diluted with 125 mL 20mM sodium acetate (NaAc) and pH 6 confirmed. If necessary, pH wasadjusted with HCl.

Analysis was performed on an Agilent 1100 series HPLC using a Dionex4×250 mm DNAPak column. Buffer A was 1 mM EDTA, 25 mM Tris pH 9, 50 mMNaClO₄, 20% MeCN. Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO₄,20% MeCN. Separation was performed on a 0-65% B segmented gradient withbuffers and column heated to 65° C.

Materials were purified on an ÅKTA Pilot equipped with a FineLine-70column packed with TSKgel Q 5PW (Tosoh Biosciences) to a bed height of28 cm (=1.08 L) Buffer A was 1 mM EDTA, 25 mM Tris pH 9. Buffer B was 1mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO₄. Buffers were heated by a 4 kWbuffer heater set at 65° C., giving a column outlet temperature of 45°C. The solution containing the crude material was diluted 4-6 fold andloaded onto the column at 200 mL/min and eluted with a segmentedgradient from 0-60% B. Appropriate fractions were pooled and this pooledmaterial desalted in 30 mL amounts over Sephadex G-25 on a BioPilotcolumn (6 cm dia.×7.5 cm) against H₂O. The eluate was vacuum evaporatedto less than 25 mL, shell frozen and lyophilized.

The results of the synthesis of 25 and 26 with PADS or EDITH are shownin FIG. 6. It should be noted that the contact time used for EDITH isless than that suggested by Q. Xu et al. (one vs. two minutes.)

Example 5 Deprotection Conditions General

The following oligonucleotide sequences used for various deprotectionmethods.

27: 5′CUUACGCUGAGUACUUCGAdTdT P═O RNA (SEQ ID NO: 8) 28:5′UCGAAGUACUCAGCGUAAGdTdT. P═O/P═S RNA (SEQ ID NO: 9) 29:5′GCGGAUCAAACCUCACCAAdTdT. P═O backbone (SEQ ID NO: 10) 30:5′GCGGAUCAAACCUCACCAAdTdT. P═O/P═S mixed backbone (SEQ ID NO: 11) 31:5′GCGGAUCAAACCUCACCAAdTdT. P═S backbone (SEQ ID NO: 12) 32:5′UUGGUGAGGUUUGAUCCGCdTdT. P═O backbone (SEQ ID NO: 13) 33:5′UUGGUGAGGUUUGAUCCGCdTdT. P═O/P═S mixed backbone (SEQ ID NO: 14) 34:5′UUGGUGAGGUUUGAUCCGCdTdT. P═S backbone (SEQ ID NO: 15)

Method 1

A volumetric mixture (˜1:4) of Py.HF and DBU with DMSO (4-5 volume ofPyHF) as solvent at 65° C. for 15 mins. This is a two step reactioncondition.

Control: A ˜1 umole sample of 27 was deprotected by MeNH₂ at 65° C. for20 mins and dried. Then it was treated with a mixture of 0.1 mL TEA.3HF,0.075 mL TEA and 0.15 mL DMSO at 65° C. for 1.5 hours. The yield on HPLCwas 47/54% (260 nm and 280 nm) on anion exchange HPLC. A 0.5 mmole ODsample of dried 27, deprotected by MeNH₂ at 65° C. for 20 mins, wasdissolved in premixed 10 μL Py.HF, 50 μL DBU and 50 μL DMSO and heatedat 65° C. The yield was 55/53% after 10 mins, 57/57% after 20 mins,57/58% after 30 mins and 57/57% after 1 hour. The pH of this 1:5 mixturewas found out to be about 10 by adding in water. Therefore, ˜0.5 mmoleof the MeNH₂ deprotected and dried 27 was deprotected by premixed 6.5 μLPy.HF, 27.4 μL DBU and 26 μL DMSO at 65° C. for 15 mins and 70 mins. Theyield was 57/57% after 15 mins and 70 mins. A ˜4 μmole sample of 27 wasdeprotected by concentrated ammonia at 65° C. for 1 hour and dried. Theresidue was then dissolved in premixed 0.06 mL Py.HF, 0.24 mL DBU, and0.3 mL DMSO at 65° C. for 15 mins. The yield was 58/60%. A ˜4 μmolesample of 27 was deprotected by ethanolic ammonia at 65° C. for 1 hourand dried. Premixed 0.06 mL Py.HF, 0.24 mL DBU, and 0.3 mL DMSO wereused to treat the RNA at 65° C. for 15 min. The yield was 59/60%.

Compound 29 was synthesized at 1 mmole scale. It was deprotected byethanolic ammonia at 65° C. for 1 hour, then divided to half (71 OD and77 OD) and dried. 27 μL Py.HF, 108 μL DBU and 135 μL DMSO were mixed.Half of this mixture was used to treat the 77 OD sample for 20 mins at65° C., the other half was used to treat the 71 OD sample for 30 mins.The yield was 64/63% after 20 mins and 62/63% after 30 mins. The fullythioated 31 was deprotected by ethanolic ammonia at 65° C. for 45 mins.The crude mixture was divided into half and dried, 76 OD in each sample.20 μL Py.HF, 80 μL DBU and 100 μL DMSO were premixed, half of it wereused to dissolve one sample and the other half for the other sample. At65° C., the yield was 64/81% after 20 mins and 63/81% after 30 mins. NoPS/PO conversion was detected on LC-MS.

Part of 28 was deprotected with MeNH₂ at 65° C. for 20 mins. The crudemixture was divided into ˜40 OD samples and dried. The other part wasdeprotected with ethanolic ammonia at 65° C. for 40 mins, and alsodivided into ˜40 OD samples and dried. One portion of MeNH₂ deprotectedsample was desilylated with standard procedures (16 μL TEA.3HF, 12 μLTEA and 24 μL DMSO at 65° C.), the yield was 37/36% after 30 mins,41/49% after 1 hour, 38/43% after 1.5 hours and 42/42% after 2.5 hours.Second portion of MeNH₂ deprotected sample was desilylated with premixed9 μL Py.HF, 36 μL DBU and 36 μL DMSO at 65° C., and the yield was 44/45%after 15 mins, 46/45% after 30 mins, 45/44% after 1 hour, 45/44% after1.5 hr and 44/48% after 2.5 hrs. Another portion of MeNH₂ deprotectedsample was desilylated with premixed 9 μL Py.HF, 31.5 μL DBU and 31.5 μLDMSO at 65° C., and the yield was 42/45% after 15 mins, 45/47% after 30mins, 45/44% after 1 hour, 45/48% after 1.5 hr and 39/47% after 2.5 hrs.One portion of ethanolic ammonia deprotected sample was desilylated withstandard procedures (16 μL TEA.3HF, 12 μL TEA and 24 μL DMSO at 65° C.),the yield was 40/39% after 30 mins, 49/51% after 1 hour, 49/51% after1.5 hour and 47/49% after 2.5 hour. Second portion of ethanolic ammoniadeprotected sample was desilylated with premixed 9 μL Py.HF, 36 μL DBUand 36 μL DMSO at 65° C., and the yield was 50/50% after 15 mins, 49/49%after 30 mins, 53/54% after 1 hour, 55/58% after 1.5 hour and 54/54%after 2.5 hrs. Another portion of ethanolic ammonia deprotected samplewas desilylated with premixed 9 μL Py.HF, 31.5 μL DBU and 31.5 μL DMSOat 65° C., and the yield was 52/52% after 15 mins, 52/51% after 30 mins,52/52% after 1 hour, 53/55% after 1.5 hour and 52/55% after 2.5 hour.

Standard deprotection of 29 gave 47/48% yield. Ethanolic ammoniadeprotection of 29 at 65° C. for 1 hour followed by 15 mins treatmentwith premixed 105 μL Py.HF, 367.5 μL DBU and 300 μL DMSO at 65° C. gave47/49% yield. Part of the support was treated with ethanolic ammonia for1.5 hr at 65° C. and then dissolved in premixed 105 μL Py.HF, 367.5 μLDBU and 300 μL DMSO at 65° C. for 15 mins, which gave 47/47% yield.

Deprotection for 1 hr in ethanolic ammonia at 65° C. followed by 65° C.and 20 mins/15 mins 1:3.5 mixture desilylation was applied on 32/34 gave60/61% and 61/61% yields respectively. For 33 synthesized on 1 mmolescale, both standard and Pyridine-HF/DBU deprotections were done, andyields were 41/40% for standard and 45/43% for Pyridine-HF/DBU method.

Method 2: One Step Process

Silyl deprotection reagent: 4 volume desilylation mixture (1 mL Py.HF,3.5 mL DBU, 4 mL DMSO) per 1 volume of ethanolic ammonia at 60° C. for20 mins.

This method was tested with a ˜40 OD sample of 28 after MeNH₂deprotection. 20 μL of ethanolic ammonia was used to dissolve the oligo,and then 80 μL of premixed Py.HF reagent (1 mL Py.HF+3.5 m DBU+4 mLDMSO) were added in to the sample. The yield was 49/45% when heated at60° C. for 20 mins, 1 hour and 2 hours. Under this condition thedeprotecion was complete in 20 minute without any degradation of theRNA.

Method 3: A Two Step Process.

Silyl deprotection reagent: 5 μL DMSO and 2.5 μL DBU per 1 mg ofpoly{4-vinylpyridinium poly(hydrogen fluoride)] (PVPHF) at 65° C. for 20min.

About 40 OD of dried sample of ethanolic ammonia deprotected 27 wasdissolved in 50 μL DMSO. 25 μL DBU and 10 mg PVPHF were added in andheated at 65° C. The yield was 52/51% after 20 mins, 54/57% after 40mins and 55/62% after 90 mins. When the sample was treated with 50 μLDMSO, 30 μL DBU and 10 mg PVPHF at 65° C., the yield was 48/51% after 20mins, 50/50% after 40 mins and 48/48% after 1.5 hours.

Method 4: One Step Deprotection

One-step deprotection with PVPHF: for every 10 μL ethanotic ammonia, add˜30-40 μL DMSO and 3 mg PVPHF. The deprotection takes up to 1.5 hours.

About 40 OD dried sample of ethanolic ammonia deprotected 28 wasredissolved in 30 ethanolic ammonia, and 90 μL DMSO and 9 mg PVPHF wereadded into it. The deprotection was not complete after 20 mins. Yieldwas 49/51% after 40 mins and 51/51% after 1.5 hours. A second portion of28 was redissolved in 25 μL ethanolic ammonia and 100 μL DMSO with 9 mgPVPHF. The reaction was not complete after 20 min. The yield was 41/50%after 40 min and 50/57% after 1.5 hour. When a portion of 28 deprotectedby MeNH₂ was redissolved in 20 μL ethanolic ammonia and 80 μL DMSO with10 mg PVPHF gave 42/42% yield after 50 mins.

Method 5

One-step deprotection with PVPHF: for every 10 μL ethanolic ammonia, add˜30-40 μL DMSO, 5 μL DBU and ˜4.5 mg PVPHF. The deprotection takes up to40 min.

A ˜40 OD dried sample of MeNH₂ deprotected 28 was redissolved in 20 μLethanolic ammonia, and then 80 μL DMSO, 10 μL DBU and 9 mg PVPHF wereadded into solution. This method gave 45/45% after 40 min and 46/49%yield after 1.5 hour.

Method 6: Tris(Dimethylamino)Sulfur Difluorotrimethylsilane (TAS-F) asSilyl Deprotecting Agent for RNA Synthesis

About 1 μmole methylamine deprotected and dried 27 was treated with asolution of 0.16 g TAS-F in 0.2 mL of DMF at 55° C. for 2 hours. Thereaction was not complete and the reaction mixture was not homogenouswith some gel sitting out of the solution. 20 μL water was added intothe reaction mixture. The reaction mixture became clear after overnightstoring at 55° C. HPLC purification gave 51/55% for this reaction. Thereproducibility of this reaction was not very consistent. ˜0.6 μmole of27 was treated with 80 mg TAS-F and 0.2 mL pyridine at 65° C. Only22/21% yield was observed after 2 hours. ˜0.6 μmole was treated with 80mg TAS-F and 0.2 mL N-methylpyrrolidinone at 65° C. A precipitate wasformed during the course of the reaction and the yield was 34/37% after2 hrs. ˜0.4 μmole of 27 was treated with 27 mg TAS-F, 0.15 mLN-methylpyrrolidinone and 0.5 mL DMSO at 65° C. for 2 hours. The yieldwas 35/24%˜0.4 μmole was treated with 27 mg TAS-F, 0.15 mLlN-methylpyrrolidinone and 0.05 mL DMSO at 65° C. for 2 hours. The yieldwas 25/25%. ˜0.4 μmole of 27 was treated with 27 mg TAS-F, 0.15 mLN-methylpyrrolidinone and 0.05 mL pyridine at 65° C. for 2 hours. Theyield was 22/22%. ˜1 μmole of ethanolic ammonia deprotected and dried 27was treated with 75 mg TAS-F and 0.2 mL DMSO at 65° C. The yield was39/41% after 2 hours. ˜1 μmole of this sample was treated with 75 mgTAS-F and 0.2 mL DMF at 65° C. Precipitate formed during the course ofthe reaction and the yield was 21/21% after 2 hours. ˜1 μmole of ammoniadeprotected and dried 27 was treated with 75 mg TAS-F and 0.2 mL DMSO at65° C. The yield was 31/30% after 2 hours. ˜1 μmole of this sample wastreated with 75 mg TAS-F and 0.2 mL DMF at 65° C. Precipitate formed andthe yield was 21/24% after 2 hours.

A ˜40 OD sample of MeNH₂ deprotected (65° C. 20 mins) and dried 28sample was treated with 41 mg TASF and 90 μL DMF at 65° C. Injectionswere done after 30 mins, 1 hr, 2 hr, and then at RT overnight. Thereaction did not yield noticeable amount of product. Another ˜40 ODsample was treated with 41 mg TASF, 90 μL DMF and 40 μL water at 65° C.Injections were done after 30 min, 1 hr, 2 hr, and then at RT overnight.No major peak was detected in the HPLC for the product. Samedeprotection conditions were applied on ˜40 OD samples of 28 deprotectedby ethanolic ammonia (65° C., 40 min.) and same results were observed:no major peak.

Example 6 Microwave-Mediated Deprotection of a 2′-Silyl Group of RNA A.Deprotection 1 (Standard)

The oligonucleotide was cleaved from the support with simultaneousdeprotection of base and phosphate groups with 2.0 mL of a mixture ofammonia and 8 M ethanolic methylamine [1:1] for 30 min at 65° C. Thevial was cooled briefly on ice and then the ethanolic ammonia mixturewas transferred to a new microfuge tube. The CPG was washed with 2×0.1mL portions of deionized water, put in dry ice for 10 min, and thendried in speed vac.

B. Microwave Deprotection of 2′-O-TBDMS Group of RNA

About 12 OD of Oligo 50 or 51 was resuspended in 600 μL of Reagent A toC. The vial containing the oligonucleotides was then placed in microwaveunit. The solution was irradiated for 2 min. and 4 min. in CEM DiscoverExplorer.

Work Up

Condition A: In case of TBAF after Microwave irradiation quenched thereaction with water followed by desalting.

Condition B: The reaction was then quenched with 400 μL ofisopropoxytrimethylsilane (iPrOSiMe₃, Aldrich) and further incubated onthe heating block leaving the caps open for 10 min. (This causes thevolatile isopropxytrimethylsilylfluoride adduct to vaporize). Theresidual quenching reagent was removed by drying in a speed vac. Added1.5 mL of 3% triethylamine in diethyl ether and pelleted bycentrifuging. The supernatant was pipetted out without disturbing thepellet. Dry the pellet in speed vac. The crude RNA was obtained as awhite fluffy material in the microfuge tube.

Microwave deprotection RNA and its MS Analysis 2′-silyl Com-deprotection cal. found pound Sequence condition mass mass 50 5′ACGUCGAUAT 3′ TBAF 2 min 3142.95 3142.57 (SEQ ID NO: 16) 50 5′ACGUCGAUAT 3′ Py.HF 2 min 3142.95 nd (SEQ ID NO: 16) 50 5′ ACGUCGAUAT 3′Py.HF 4 min 3142.95 nd (SEQ ID NO: 16) 51 5′ CGUCAAGGCGAT 3′ TBAF 2 min3832.37 3831.34 (SEQ ID NO: 17) 51 5′ CGUCAAGGCGAT 3′ TEA.3HF 2 min3832.37 3831.34 (SEQ ID NO: 17) 51 5′ CGUCAAGGCGAT 3′ TEA.3HF 4 min3832.37 3831.34 (SEQ ID NO: 17) nd: not determined

Example 7

The Applicants have surprisingly discovered that impurities in acomposition of single stranded RNA can be readily removed by HPLCpurification of a mixture of single-stranded RNA that has been annealedto generate double-stranded RNA.

General Procedure

A diagram illustrating the overall purification procedure is presentedin FIG. 9. The specific procedure used for the purification ofAL-DP-4014 is presented in FIGS. 11 and 12.

The analytical conditions used for reverse phase HPLC purification, ionexchange purification, capillary gel electrophoresis, and LC-MS arepresented below.

Reverse Phase HPLC:

Luna C-18 column, 150×2.0 mm, temp=25° C., flow=0.2 mL/min

Buffer A: 35 mm TEAA PH=7, 100 mm HFIP

Buffer B: MeOH

Gradient: 25% B to 35% B in 50 minutes, ramp to 85% B at 55 minutes,re-equilibrate

Ion Exchange Chromatography:

Dnapac PA-100 ion exchange column, 250×4 mm, temp=65° C., flow=1 ml/min

Buffer A: 50 mm NaClO₄, 25 mm tris pH=9.0, 1 MM EDTA, 20% CAN

Buffer B: 400 mm NaClO₄, 25 mm tris pH=9.0, 1 MM EDTA, 20% CAN

Gradient: hold at 0% B for 2.00 min, ramp to 40% B at 17 min, ramp to65% B at 32 min, ramp to 100% B at 32.5 min. re-equilibrate

Capillary Gel Electrophoresis:

DNA 100R Gel, temp=40° C.

Separate at 12 KV, reverse polarity

LC-MS Analysis:

Chromolith speedrod 50×4 mm temp=25° C., flow=0.8 mL/min

Buffer A: 20% MeOH, 10 mm TBAA pH=7.0

Buffer B: 80% MeOH, 10 mm TBAA pH=7.0

Gradient: 40% B to 80% B in 19.5 min., ramp to 100% B at 23 minutesre-equilibrate Scan MS in negative ion mode from 500 to 3000

Results

The specific procedure used for the purification of AL-DP-4014 ispresented in FIGS. 11 and 12. The chromatographic data presented inFIGS. 14-18 indicate that the purification procedure produced AL-DP-4014in substantially pure form. The purification procedure was performed asdescribed above for AL-DP-4127, AL-DP-4139, AND AL-DP-414. The resultsfrom analytical analyses are presented in FIGS. 19-39.

Example 8 Procedure for Quenching Acrylonitrile

The solid support bound oligonucleotide is treated with exceess of amixture of triethylamine (or an amine with pKa=9-12), an organic solvent(e.g. acetonitrile, THF) and a thiol or a odorless thiol. The alkylaminewould generate the acrylonitlile which would be scavenged by the thiol.This is an improvement over the process described by Capaldi et al. Org.Process Res. Dev. 2003, 7, 832-838.

Example 9 2′-O-Methyl-Modified, 2′-Fluoro-Modified, Conjugated, ThioateOligonucleotides Step 1. Oligonucleotide Synthesis

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid supports (dT-CPG,rC-CPG, rU-CPG, from Prime Synthesis) or the in-house synthesized solidsupports (phthalimido-hydroxy-prolinol-CPG,hydroxyprolinol-cholesterol-CPG described in patent applications:provisional 60/600,703 Filed Aug. 10, 2004 and PCT/US04/11829 Filed Apr.16, 2004) were used for the synthesis. RNA phosphoramidites and2′-O-methyl modified RNA phosphoramidites with standard protectinggroups(5′-O-dimethoxytrityl-N-6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N6-benzoyl-2′-O-methyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-O-methyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-O-methyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite)were obtained from Pierce Nucleic Acids Technologies and ChemGenesResearch. The 2′-F phosphoramidites(5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite)were obtained from Promega. All phosphoramidites were used at aconcentration of 0.2 M in CH₃CN except for guanosine and2′-O-methyl-uridine, which were used at 0.2 M concentration in 10%THF/CH₃CN (v/v). Coupling/recycling time of 16 minutes was used for allphosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole(0.75 M, American International Chemicals). For the PO-oxidation, 50 mMiodine in water/pyridine (10:90 v/v) was used and for the PS-oxidation2% PADS (GL Synthesis) in 2,6-lutidine/CH₃CN (1:1 v/v) was used. Thecholesterol and amino-linker phosphoramidites were synthesized in house,and used at a concentration of 0.1 M in dichloromethane for cholesteroland 0.2 M in CH₃CN for the amino-linker. Coupling/recycling time forboth the cholesterol and the amino-linker phosphoramidites was 16minutes.

Step 2. Deprotection of Oligonucleotides

(a) Deprotection of RNAs without the 2′-fluoro modification: Aftercompletion of synthesis, the support was transferred to a 100 mL glassbottle (VWR). The oligonucleotide was cleaved from the support withsimultaneous deprotection of base and phosphate groups with 40 mL of a40% aq. methyl amine (Aldrich) 90 mins at 45° C. The bottle was cooledbriefly on ice and then the methylamine was filtered into a new 500 mLbottle. The CPG was washed three times with 40 mL portions of DMSO. Themixture was then cooled on dry ice.

In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′position, 60 mL triethylamine trihydrofluoride (Et₃N—HF) was added tothe above mixture. The mixture was heated at 40° C. for 60 minutes. Thereaction was then quenched with 220 mL of 50 mM sodium acetate (pH 5.5)and stored in the freezer until purification.

(b) Deprotection of 2′-fluoro modified RNAs: After completion ofsynthesis, the support was transferred to a 100 mL glass bottle (VWR).The oligonucleotide was cleaved from the support with simultaneousdeprotection of base and phosphate groups with 80 mL of a mixture ofethanolic ammonia (ammonia:ethanol, 3:1 v/v) for 6.5 h at 55° C. Thebottle was cooled briefly on ice and then the ethanolic ammonia mixturewas filtered into a new 250 mL bottle. The CPG was washed with twicewith 40 mL portions of ethanol/water (1:1 v/v). The volume of themixture was then reduced to ˜30 mL by roto-vap. The mixture was thenfrozen on dry ice and dried under vacuum on a speed vac.

The dried residue was resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (Et₃N.3HF), and DMSO (3:4:6) and heatedat 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS)groups at the 2′ position. The reaction was then quenched with 50 mL of20 mM sodium acetate and the pH was adjusted to 6.5, and the solutionwas stored in freezer until purification.

Step 3. Quantitation of Crude Oligonucleotides

For all samples, a 10 μL aliquot was diluted with 990 μL of deionisednuclease free water (1.0 mL) and the absorbance reading at 260 nm wasobtained.

Step 4. Purification of Oligonucleotides

(a) Unconjugated oligonucleotides: The unconjugated crudeoligonucleotides were first analyzed by HPLC (Dionex PA 100). Thebuffers were 20 mM phosphate, pH 11 (buffer A); and 20 mM phosphate, 1.8M NaBr, pH 11 (buffer B). The flow rate 1.0 mL/min and monitoredwavelength was 260-280 nm. Injections of 5-15 μL were done for eachsample.

The unconjugated samples were purified by HPLC on an TSK-Gel SuperQ-5PW(20) column packed in house (17.3×5 cm). The buffers were 20 mMphosphate in 10% CH₃CN, pH 8.5 (buffer A) and 20 mM phosphate, 1.0 MNaBr in 10% CH₃CN, pH 8.5 (buffer B). The flow rate was 50.0 mL/min andwavelengths of 260 and 294 nm were monitored. The fractions containingthe full-length oligonucleotides were pooled together, evaporated, andreconstituted to about 100 mL with deionised water.

(b) Cholesterol-conjugated oligonucleotides: The cholesterol-conjugatedcrude oligonucleotides were first analyzed by LC/MS to determine purity.The 5′-cholesterol conjugated sequences were HPLC purified on anRPC-Source 15 reverse-phase column packed in house. The buffers were 20mM TEAA in 10% CH₃CN (buffer A) and 20 mM TEAA in 70% CH₃CN (buffer B).The fractions containing the full-length oligonucleotides were thenpooled together, evaporated, and reconstituted to 100 mL with deionisedwater. The 3′-cholesterol conjugated sequences were HPLC purified on anRPC-Source 15 reverse-phase column packed in house. The buffers were 20mM NaOAc in 10% CH₃CN (buffer A) and 20 mM NaOAc in 70% CH₃CN (bufferB). The fractions containing the full-length oligonucleotides werepooled, evaporated, and reconstituted to 100 mL with deionised water.

Step 5. Desalting of Purified Oligonucleotides

The purified oligonucleotides were desalted on an AKTA Explorer system(Amersham Biosciences) using a Sephadex G-25 column. First, the columnwas washed with water at a flow rate of 25 mL/min for 20-30 min. Thesample was then applied in 25 mL fractions. The eluted salt-freefractions were combined, dried, and reconstituted in 50 mL of RNase freewater.

Step 6. Purity Analysis by Capillary Gel Electrophoresis (CGE),Ion-Exchange HPLC, and Electrospray LC/Ms

Approximately 0.3 OD of each of the desalted oligonucleotides werediluted in water to 300 μL and were analyzed by CGE, ion exchange HPLC,and LC/MS.

Calc Found Purity AL-SQ # Sequence Target Mass Mass (%) 2936 HP-NH2- Luc6915 6915.01 97.8* CUUACGCUGAGUACUUCGAdTsdT (SEQ ID NO: 18) 2937CsUUACGCUGAGUACUUCGAdTdTdT-HP-NH2 Luc 6915 6915.06 95.9* (SEQ ID NO: 19)5225 GUCAUCACACUGAAUACCAAUs-Chol ApoB 7344 7344.70 83 (SEQ ID NO: 20)3169 U_(F) _(S) U_(F)GGAUC_(F)AAAU_(F)AU_(F)AAGAU_(F)UCC_(F) _(S) C_(F)_(S) U ApoB 7325.39 7325.5 92 (SEQ ID NO: 21) 2920GGAC_(F)U_(F)AC_(F)U_(F)C_(F)U_(F)AAGU_(F)U_(F)C_(F)U_(F)AC_(F)dTsdTFactor VII 6628.93 6628.45 99.6 (SEQ ID NO: 22) 2921GU_(F)AGAAC_(F)U_(F)U_(F)AGAGU_(F)AGU_(F)C_(F)C_(F)dTsdT Factor VII6726.04 6725.78 96.0 (SEQ ID NO: 23) 4723GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGU_(F)C_(F)U_(F)U_(F)AC_(F)dTsdTFactor VII 6628.93 6628.47 98.9 (SEQ ID NO: 24) 4724GU_(F)AAGAC_(F)U_(F)U_(F)GAGAU_(F)GAU_(F)C_(F)C_(C)dTsdT Factor VII6726.04 6725.56 96.3 (SEQ ID NO: 25) 3000CsGUCU_(F)GUCU_(F)GUCCCGGAUCdTsdT G6P 6610.94 6611.34 92 (SEQ ID NO: 26)3002 GsAUCCGGGAC_(F)AGAC_(F)AGACGdTsdT G6P 6806.2 6806.06 93(SEQ ID NO: 27) 2918 Chol- Factor VII 7332.93 7333.61 99.9GGAU_(F)C_(F)AU_(F)C_(F)U_(F)C_(F)AAGU_(F)C_(F)U_(F)U_(F)AC_(F)dTsdT(SEQ ID NO: 28) 2919 Chol- Factor VII 7332.93 7333.62 99.6GGAC_(F)U_(F)AC_(F)U_(F)C_(F)U_(F)AAGU_(F)U_(F)C_(F)U_(F)AC_(F)dTsdT(SEQ ID NO: 29) 3168GsGAAUCU_(F)U_(F)AU_(F)AU_(F)U_(F)U_(F)GAUCC_(F)AAs-Chol ApoB 73937393.3 76.4 (SEQ ID NO: 30) 3001 CsGUCU_(F)GUCU_(F)GUCCCGGAUCdTsdTs-CholG6P 7330.94 7331.3 79.4 (SEQ ID NO: 31) 4968CGUCCU_(OMe)GAAGAAGAU_(OMe)GGU_(OMe)GC_(OMe)sG_(OMe)sC GFP 7504.77504.20 92 (SEQ ID NO: 32) 5226 AUUGGUAUUCAGUGUGAUGAC_(OMe)sA_(OMe)sCApoB 7409.5 7409.80 91 (SEQ ID NO: 33) 5475U_(OMe)U_(OMe)GGAU_(OMe)AAAU_(OMe)AU_(OMe)AAGAU_(OMe)UCC_(OMe)sC_(OMe)sUApoB 7421.7 7421.4 89 (SEQ ID NO: 34) 3196CsU_(OMe)AUGAGCCUGAAGCC_(OMe)U_(OMe)A_(OMe)AdTsdT a-synuclein 6741.26741.01 92.6 (SEQ ID NO: 35) 3197U_(OMe)sU_(OMe)AGGCUUCAGGCUCAU_(OMe)AGdTsdT a-synuclein 6721.12 6720.9391.9 (SEQ ID NO: 36) 3199CsU_(OMe)ACGAACCUGAAGCC_(OMe)U_(OMe)A_(OMe)AdTsdT a-synuclein 6724.216723.94 92.2 (SEQ ID NO: 37) 3200U_(OMe)sU_(OMe)AGGCUUCAGGUUCGU_(OMe)AGdTsdT a-synuclein 6738.11 6737.8879.6 (SEQ ID NO: 38) 3201CsU_(OMe)ACGAACCUGAAGCC_(OMe)U_(OMe)A_(OMe)AdTsdTs-Chol a-synuclein7444.21 7445.08 91.4 (SEQ ID NO: 39) 3198CsU_(OMe)AUGAGCCUGAAGCC_(OMe)U_(OMe)A_(OMe)AdTsdTs-Chol a-synuclein7461.2 7462.02 85.7 (SEQ ID NO: 40) 3131AsGAAGC_(OMe)AGGACCUU_(OMe)AUCU_(OMe)AdTsdTs-Chol ApoB 7471.1 7472.1797.6 (SEQ ID NO: 41) 5474GGAAUCU_(OMe)U_(OMe)AU_(OMe)AU_(OMe)U_(OMe)U_(OMe)GAUCC_(OMe)AAs-CholApoB 7461.1 7461.9 83 (SEQ ID NO: 42) 4967GC_(OMe)ACC_(OMe)AUCUUCUUC_(OMe)AAGGACGs-Chol GFP 7394 7394.80 91(SEQ ID NO: 43) 3037A_(OMe)sC_(OMe)sA_(OMe)sA_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sC_(OMe)sA_(OMe)smiR-122A 8613.43 8614.53 82.7U_(OMe)sU_(OMe)sG_(OMe)sU_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sU_(OMe)sC_(OMe)sC_(OMe)sA_(OMe)s-Chol (SEQ ID NO: 44) 3038A_(OMe)sC_(OMe)sA_(OMe)sA_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sC_(OMe)sA_(OMe)smiR-122A 8340.09 8341.23 99.2U_(OMe)sU_(OMe)sG_(OMe)sU_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sU_(OMe)sC_(OMe)sC_(OMe)sA_(OMe)s-Chol (SEQ ID NO: 45) 3039A_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)smiR-122A 8613.43 8614.75 86.6C_(OMe)sU_(OMe)sG_(OMe)sU_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sU_(OMe)sU_(OMe)sC_(OMe)sC_(OMe)sA_(OMe)s-Chol (SEQ ID NO: 46) 3040A_(OMe)sC_(OMe)sA_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)smiR-122A 8340.09 8341.15 85.2U_(OMe)sG_(OMe)sU_(OMe)sC_(OMe)sA_(OMe)sC_(OMe)sA_(OMe)sU_(OMe)sU_(OMe)sC_(OMe)sC_(OMe)sA_(OMe)s-Chol (SEQ ID NO: 47)The strands are shown written 5′ to 3′. Lower case “s” indicates aphosphorothioate linkage. The lower case “d” indicates a deoxy residue.“HP—NH2” or “NH2-HP” indicates a hydroxyprolinol amine conjugate.“Chol-” indicates a hydroxyprolinol cholesterol conjugate. Subscript“OMe” indicates a 2′-O-methyl sugar and subscript “F” indicates a2′-fluoro modified sugar. Purity was determined by CGE except whereindicated by an asterisk (in these two cases, purity was determined byion-exchange chromatography).

Example 10 Deprotection Methods of RNA (with 2′-OMe, PS, or CholesterolModifications) Using Py.HF and Polyvinylpyridine polyHF (PVPHF) Step 1.Oligonucleotide Synthesis

All oligonucleotides were synthesized on an AKTA oligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG,U-CPG 500 {acute over (Å)}) or the hydroxy-prolinol-cholesterol solidsupport (described in patent application s: provisional 60/600,703 FiledAug. 10, 2004 and PCT/US04/11829 Filed Apr. 16, 2004) was used. RNAphosphoramidites with standard protecting groups,5′-O-dimethoxytrityl-N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditeand5′-O-dimethoxytrityl-thymidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditewere used for the oligonucleotide synthesis. All phosphoramidites wereused at a concentration of 0.2 M in acetonitrile (CH₃CN) except forguanosine and 2′-OMe uridine which was used at 0.2 M concentration in10% THF/acetonitrile (v/v). Coupling/recycling time was 14 minutes withlinear flow of 500 cm/h on a 12 mL synthesis column. The activator was5-ethyl thiotetrazole (0.75M). For the PO-oxidation 0.5 M iodine inpyridine with 10% water was used and for the PS-oxidation 0.2 M PADS in1:1 mixture of CH₃CN and 2,6-lutidine was used. Capping mixture A was20% N-methyl imidazole and 80% CH₃CN and capping mixture B was 25%acetic anhydride, 30% 2,6-lutidine and 45% CH₃CN.

The oligonucleotides synthesized, scale, support type, amount andloading are listed below:

Alnylam Support, Mass of Loading Scale Synthesis SQ No. Mass (gram)support (g) (μmol/g) (umol) column 5718 dT 4.15 84 349 12 mL 5719 dT4.15 84 349 12 mL 3216 dT 4.01 87 349 12 mL 3218 dT 4.08 87 355 12 mL5474 Hydroxy 4.1 68.6 281 12 mL prolinol cholesterol 5475 rU 3.9 83 32412 mL

Step 2. Deprotection

Four methods of deprotection were employed to achieve the following twosteps of cleavage and deprotection: Step 1) cleavage of oligonucleotidefrom support with simultaneous removal of base and phosphate protectinggroups from the oligonucleotide, Step 2) deprotection of 2′-O-TBDMSgroups.

(a) Deprotection with Pyridine HF: The solid support from a 200 μmolsynthesis was treated with 30 mL (1 vol) of MeNH₂ (40%, aqueous) at 45°C. for 1.5 hours. The support was filtered out and rinsed with 60 mL (2vol) DMSO. Cool it for about 10 minutes in dry ice, a mixture of 7.5 mLpyridine HF (70%) and 30 mL (1 vol) DMSO was added to the filtrate andrinse solution and it was heated at 40° C. for 1 hour. The reaction wasquenched with 50 mM sodium phosphate (pH 5.5) and diluted with water toan appropriate volume.

(b) Deprotection with Pyridine HF with DBU: The solid support from a 200μmol synthesis was treated with 20 mL MeNH₂ (40%, aqueous) at 45° C. for1.5 hours. The support was filtered out and rinsed with 60 mL DMSO. 10mL DBU was added in the solution. Cool it for about 10 minutes in dryice, a mixture of 6 mL pyridine HF (70%) and 20 mL DMSO were added tothe filtrate and rinse solution and it was heated at 40° C. for 1 hour.The reaction was quenched with 50 mM sodium phosphate (pH 5.5) anddiluted with water.

(c) Deprotection with Polyvinylpyridine polyHF (PVPHF): The solidsupport from a 200 μmol synthesis was treated with 30 mL MeNH₂ (40%,aqueous) at 45° C. for 1.5 hours. The support was filtered out andrinsed with 90 mL DMSO. Cool it for about 10 minutes in dry ice, PVPHF(12 g) was added to the filtrate and rinse solution and it was heated at40° C. for 1 hour. The reaction was quenched with 50 mM sodium phosphate(pH 5.5). The reaction mixture was filtered and the solid was rinsedwith water.

(d) Deprotection with Polyvinylpyridine polyHF (PVPHF) with DBU: Thesolid support from a 200 μmol synthesis was treated 20 mL MeNH₂ (40%,aqueous) at 45° C. for 1.5 hours. The support was filtered out andrinsed with 80 mL DMSO. 8 mL DBU was added in the solution. Cool it forabout 10 minutes in dry ice, 12 g PVPHF were added into the filtrate andrinse solutions and the reaction was heated at 40° C. for 1 hour. Thereaction was quenched with 50 mM sodium phosphate (pH 5.5). The reactionmixture was filtered and the solid was rinsed with water.

Step 3. Purification of Oligonucleotides

(a) Ion Exchange HPLC Purification: The buffers used for the ionexchange purification were 20 mM sodium phosphate, 10% CH₃CN, pH 8.5(solvent A) and 20 mM sodium phosphate, 1 M NaBr, 10% CH₃CN, pH 8.5(solvent B). When the amount of crude oligonucleotide was less than10,000 OD, a Waters 2 cm column with TSK Gel super Q-5PW resin was used.The flow rate was 10 mL/min and the gradient was 0 to 20% solvent B over30 minutes, then 20 to 50% B over 200 minutes.

When the amount of crude oligonucleotide was more than 10,000 OD orhigher resolution was needed due to contamination with shortoligonucleotides, a Waters 5 cm column with TSK-GEL super Q-5PW resinwas used. The flow rate was 50 mL/min and the gradient was 0 to 20%solvent B over 30 minutes and then 20 to 50% solvent B over 200 minutes.

(b) Reverse phase HPLC Purification: For reverse phase purification, thebuffers were 20 mM sodium acetate, 10% CAN, pH 8.5 (solvent A) and 20 mMsodium acetate, 70% CH₃CN, pH 8.5 (solvent B). A 5 cm Waters column withsource 15 RPC was used. The flow rate was 50 mL/min and the gradient was0 to 15% solvent B over 30 minutes followed by 15 to 50% solvent B over160 minutes.

Step 4. Desalting of Purified Oligomer

The purified oligonucleotides were desalted on a Waters 5 cm column withsize exclusion resin Sephadex G-25. The flow rate was 25 mL/min. Theeluted salt-free fractions were combined together, dried down andreconstituted in RNase-free water.

Step 5. Capillary Gel Electrophoresis (CGE) and Electrospray LC/Ms

Approximately 0.15 OD of oligonucleotide was diluted in water to 150 μL.Mass of the product and purity (as shown below) were determined by LC/MSanalysis and anion exchange HPLC or CGE.

AL-SQ Cal. Obs. Purity Deprotect. # Target Sequence Mass Mass % Method5718 RSV GGCUCUUAGCAAAGUCAAGdTdT 6693 6693 95 Pyridine HF(SEQ ID NO: 48) 5718 RSV GGCUCUUAGCAAAGUCAAGdTdT 6693 6693 97 Pyridine(SEQ ID NO: 49) HF with DBU 5719 RSV CUUGACUUUGCUAAGAGCCdTdT 6607 660695 Pyridine HF (SEQ ID NO: 50) 5719 RSV CUUGACUUUGCUAAGAGCCdTdT 66076606 96 Pyridine (SEQ ID NO: 51) HF with DBU 3216 Apo BGGAAUCU_(OMe)U_(OMe)AU_(OMe)AU_(OMe)U_(OMe)U_(OMe)GAUCC_(OMe)AdT 67166717 93 PVPHF (SEQ ID NO: 52) 3216 Apo BGGAAUCU_(OMe)U_(OMe)AU_(OMe)AU_(OMe)U_(OMe)U_(OMe)GAUCC_(OMe)AdT 67166717 94 PVPHF (SEQ ID NO: 53) with DBU 3218 Apo BGsGAAUCUUAUAUUUGAUCCAsdT 6650 6651 PVPHF (SEQ ID NO: 54) 3218 Apo BGsGAAUCUUAUAUUUGAUCCAsdT 6650 6651 PVPHF (SEQ ID NO: 55) with DBU 5474Apo BGGAAUCU_(OMe)U_(OMe)AU_(OMe)AU_(OMe)U_(OMe)U_(OMe)GAUCC_(OMe)A_(OMe)As-Chol7461 7462 90 Pyridine (SEQ ID NO: 56) HF with DBU 5475 Apo BU_(OMe)U_(OMe)GGAUC_(OMe)AAAU_(OMe)AU_(OMe)AAGAU_(OMe)UCC_(OMe)sC_(OMe)sU7421 7421 93 Pyridine (SEQ ID NO: 57) HF with DBUOligonucleotides are shown written 5′ to 3′. Lower case “s” indicates aphosphorothioate linkage. The lower case “d” indicates a deoxy residue.Subscript “OMe” indicates a 2′-O-methyl sugar. “Chol-” indicates ahydroxyprolinol cholesterol conjugate.

Example 11 Deprotection Methods of Chimeric RNA with 2′-FluoroModification Using Polyvinylpyridine polyHF (PVPHF) Step 1.Oligonucleotide Synthesis

Synthesis, purification and desalting were same as described in Example9, Step 1.

Step 2. Deprotection

After the synthesis was completed, ˜30 mL of 0.5 M piperidine in CH₃CNwere pumped through the column at a flow rate of between 5 and 10 mL/minto remove the cyanoethyl protecting groups from phosphate linkages whilethe RNA was still attached to the support. Then, two methods ofdeprotection were evaluated to achieve the following two steps ofcleavage and deprotection: Step 1) cleavage of oligonucleotide fromsupport with simultaneous removal of base protecting groups from theoligonucleotide and Step 2) deprotection of 2′-O-TBDMS groups

(a) Deprotection with Polyvinylpyridine polyHF (PVPHF): The solidsupport from a 200 mmol synthesis was treated with 50 mL solution ofNH₃:ethanol (3:1) at 55° C. for 6 hours. The support was separated fromsolution by filtering and was rinsed with 90 mL DMSO. The solid supportwas removed by filtering. The filtrate and rinse solution was cooled forabout 10 minutes in dry ice, PVPHF (12 g) was added, and the solutionwas heated at 40° C. for 2 hours. Deprotection status was checked after1 hour, 1.5 hours, and 2 hours. The reaction was quenched with 50 mMsodium phosphate (pH 5.5). The reaction mixture was filtered and thesolid was rinsed with water.

(b) Deprotection with Polyvinylpyridine polyHF (PVPHF) with DBU: Thesolid support from a 200 μmol synthesis was treated 35 mL MeNH₂ (40%,aqueous) at 55° C. for 6 hours. The support was filtered out and rinsedwith 140 mL DMSO. DBU (7 mL) was added to the filtrate and rinsesolution. The solution was cooled for about 10 minutes in dry ice, 12 gPVPHF was added, and the reaction was heated at 40° C. for 2 hour.Deprotection status was checked after 1 hour, 1.5 hours, and 2 hours.The reaction was quenched with 50 mM sodium phosphate (pH 5.5). Thereaction mixture was filtered and the solid was rinsed with water.

Example 12 Deprotection Method for RNA Oligonucleotides Step 1.Oligonucleotide Synthesis

Synthesis, purification and desalting were same as described in ExampleX, Step 1. The synthesis of oligonucleotides AL-SQ-5548 (5′-AAA GUG CACAAC AUU AUA CdTdT-3′ SEQ ID NO: 58, where all residues were ribo exceptfor the two 3′ terminal nucleotides which were deoxy thymidine) andAL-SQ-5549 (5′-GUA UAA UGU UGU GCA CUU UdTdT-3′ SEQ ID NO: 59) was doneat 400 μmole scale. The calculated mass of AL-SQ-5548 was 6645.03; theobserved mass was 6644.94. The calculated mass of AL-SQ-5549 was6609.88; the observed mass was 6609.70.

Step 2. Deprotection Conditions

The deprotection was done at 94 mmole scale. Dried CPG (1.5 g) wasplaced in a 100 mL Schott bottle. Methyl amine (40% aqueous, 25 mL) wasadded to the bottle and the mixture was placed in a shaker oven at 45°C. for 1.5 h. The mixture was cooled and filtered into a 250 mL Schottbottle. The CPG was washed three times with 25 mL DMSO in a funnel. Thecombined filtrates were cooled for 10 min in dry ice. HF in pyridine(Aldrich, 20 mL) was added to the bottle. The mixture was shaken welland placed in a shaker oven at 40° C. for 1 h. The mixture was cooled toroom temperature and the reaction was quenched by adding 150 mL of 50 mMsodium acetate. The final solution was stored at 4° C.

Step 3. Quantitation of Crude Oligonucleotides

In order estimate the crude yield the following procedure was used.Since the pyridine present in the crude oligonucleotide solution absorbsat 254 nm, the absorbance was measured at 280 nm. A small amount of thecrude support was subjected to deprotection using TEA.3HF instead of HFin pyridine. Absorbance was measured for this sample at 254 nm and 280nm. Based on the ratio of A₂₅₄ to A₂₈₀ of this sample, the absorbance at254 nm for the sample containing pyridine was estimated.

The amount of full-length product was determined by anion exchange HPLC.For AL-SQ-5548, the full-length product was 73% of the total strandconcentration and for AL-SQ-5549 full-length product was 67%. The crudeyield was 143 OD/μmole.

Example 13 Synthesis and Deprotection Conditions for RNAs at 1.6 mmolScale Step 1. Oligonucleotide Synthesis

The oligonucleotides were synthesized on an AKTA oligopilot synthesizer.Commercially available controlled pore glass solid supports (from PrimeSynthesis) were used. RNA phosphoramidites and 2′-O-methyl modified RNAphosphoramidites with standard protecting groups(5′-O-dimethoxytrityl-N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N6-benzoyl-2′-O-methyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-O-methyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-O-methyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite)were obtained from Pierce Nucleic Acids Technologies and ChemGenesResearch. The 2′-F phosphoramidites(5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite)were obtained from Promega.

All phosphoramidites were used at a concentration of 0.15 M in CH₃CN.The RNA amidite coupling/recycling time was 23 minutes and 2 equivalentsof amidite were used. DNA coupling cycle used 60% activator, 7 minrecycling, and 2.0 equivalents of phosphoramidite. A UV watch wasintroduced in the “push” step before the “recycle” step to assureconsistency in each coupling step. The activator was 0.6 Methylthiotetrazole. For the PO-oxidation, 50 mM iodine in water/pyridine(10:90 v/v) was used; 4.5 equivalents were added in 2.5 min. ForPS-oxidation, 0.2 M PADS in acetonitrile:2,6-lutidine (1:1) was usedwith 2-5 column volumes of thiolation reagent used. The Cap A solutionwas 20% 1-methylimidazole in acetonitrile. Cap B was aceticanhydride:2,6-lutidine:acetonitrile (25:30:45). For capping, 1.5 columnvolumes were added in 1.5 min.

Step 2. Deprotection Conditions

The CPG was mixed with 180 mL of aqueous methylamine (Aldrich) in a 250mL Schott bottle. The mixture was placed in a shaker oven at 45° C. for75 min. The mixture was cooled, filtered into a 1 L Schott bottle andthe CPG was washed three times with 160 mL of DMSO. The filtrates werecombined and cooled for 10 min in dry ice. TEA.3HF (Alfa Aesar, 270 mL)was added to the mixture. The bottle was placed in a shaker oven at 40°C. for 65 min. The mixture was cooled to room temperature and thereaction was quenched with 1 L of 50 mM sodium acetate.

Step 3. Purification of Oligonucleotides

The oligonucleotides were purified by reverse phase HPLC using a matrixof TSK-GEL, SuperQ-5PW (20) in a 5 cm×17-18 cm column. The temperaturewas maintained at 55° C. to 65° C. The buffers were 20 mM sodiumphosphate, 10% ACN v/v, pH 8.5 (buffer A) and 20 mM sodium phosphate, 1M NaBr, 10% ACN, pH 8.5 (buffer B). The flow rates was 60 mL/min. Thegradient was from 20% B to 40% B in 160 min.

The solution of crude oligonucleotide was diluted 5-fold with buffer Aand loaded directly onto the purification column using a flow rate thatloaded about 20 mg crude material (based on A₂₆₀ readings) per mL ofcolumn volume. Fractions of 50 mL were collected.

Incorporation by Reference

All of the patents and publications cited herein are hereby incorporatedby reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A process comprising the steps of: a) synthesizing a nucleic acidmolecule comprising one or more nucleotides, using a method selectedfrom the group consisting of solid phase phosphoramidite, solution phasephosphoramidite, solid phase H-phosphonate, solution phaseH-phosphonate, hybrid phase phosphoramidite, and hybrid phaseH-phosphonate-based synthetic methods; b) contacting said nucleic acidmolecule from step (a) with aqueous alkylamine, ammonia, alow-volatility amino compound, or combinations thereof, under conditionssuitable for the removal of any 2′-amino protecting groups, exocyclicamino (base) protecting groups and/or phosphate protecting groups, whichmay be individually present or absent, from said molecule; c) contactingreaction mixture having said nucleic acid molecule from step (b) withpyridine-HF, DMAP-HF, urea-HF, TSAF, DAST, polyvinyl pyridine-HF or anamine-HF reagent of formula AA

and a polar solvent under conditions suitable for the removal of a silylprotecting group and/or a 2′-OH protecting group, wherein R¹ is alkyl,aryl, heteroaryl, aralkyl or heteroaralkyl; R² is alkyl, aryl,heteroaryl, aralkyl or heteroaralkyl; R³ is aryl or heteroaryl; and n is1 to 20; d) loading reaction mixture having said nucleic acid moleculefrom step (c) onto a chromatography media in a suitable buffer; and e)applying a purification gradient using a suitable elution buffer,analyzing the fractions and allowing for the pure fractions to be pooledand desalted.
 2. The process of claim 1, wherein said nucleic acidmolecule comprises one or more ribonucleotides.
 3. The process of claim2, wherein said nucleic acid molecule is a siRNA molecule.
 4. Theprocess of claim 2, wherein said nucleic acid molecule comprises one ormore 2′-deoxy-2′-fluoro nucleotides.
 5. The process of claim 2, whereinsaid nucleic acid molecule comprises one or more deoxyribonucleotides.6. The process of claim 1, wherein said nucleic acid molecule comprisesone or more chemical modifications selected from the group consisting ofa sugar modification, a base modification, a backbone modification and aconjugation to one or more lipophilic moieties.
 7. The process of claim6, wherein said sugar modification is a 2′-sugar modification or a3′-sugar modification.
 8. The process of claim 7, wherein said 2′-sugarmodification is a 2′-O-methyl modification.
 9. The process of claim 6,wherein said backbone modification is a phosphate backbone modificationselected from the group consisting of phosphorothioate,phosphorodithioate, alkylphosphonate, thionoalkylphosphonate,phosphinate, phosphoamidate, thionophosphoramidate, boranophosphate andcombinations thereof.
 10. The process of claim 6, wherein said chemicalmodification is a conjugation to one or more lipophilic moieties, andthe conjugated lipophilic moieties comprise a cholesterol or acholesterol derivative.
 11. The process of claim 6, wherein said nucleicacid molecule comprises one or more terminal end modifications at the3′-end, 5′-end, or both the 5′- and 3′-end of the nucleic acid molecule.12. The process of claim 1, wherein said synthetic method is solid phasephosphoramidite, solution phase phosphoramidite, or hybrid phasephosphoramidite.
 13. The process of claim 1, wherein said aqueousalkylamine is aqueous methylamine.
 14. The process of claim 1, whereinsaid low-volatility amino compound is selected from the group consistingof polyamine, PEHA, PEG-NH₂, short PEG-NH₂, cycloalkyl amine,hydroxycycloalkyl amine, hydroxyamine, K₂CO₃/MeOH, thioalkylamine,thiolated amine, β-amino-ethyl-sulfonic acid or a sodium sulfatethereof, and combinations thereof.
 15. The process of claim 1, whereinsaid aqueous alkylamine, ammonia, low-volatility amino compound orcombination thereof is premixed with ethanol.
 16. The process of claim1, wherein said 2′-OH protecting group comprises thet-butyldimethylsilyl (TBDMSi) protecting group.
 17. The process of claim1, wherein said polar solvent is selected from the group consisting ofwater, DMSO, DMF, ethanol, isopropanol, methanol, acetonitrile, andcombinations thereof.
 18. The process of claim 1, wherein pyridine-HF isused in step c) and premixed with DMSO and a base selected from thegroup consisting of DBU, Hunig's base, pyridine, piperidine andN-methylimidazole.
 19. The process of claim 1, wherein polyvinylpyridine-HF is used in step c).
 20. The process of claim 1, wherein saidnucleic acid molecule is a double-stranded nucleic acid molecule. 21.The process of claim 1, wherein said nucleic acid molecule is asingle-stranded nucleic acid molecule.
 22. The process of claim 1, wheresaid chromatography media is an ion exchange chromatography media, andsaid loading buffer comprises water, ethanol, or acetonitrile.
 23. Theprocess of claim 1, further comprising the steps of: annealing saidnucleic acid molecule with a second nucleic acid molecule to form adouble-stranded nucleic acid molecule, with or without applying thedesalted step in step e); and loading said double-stranded nucleic acidmolecule onto a chromatographic purification.
 24. A process comprisingthe steps of: a) synthesizing a nucleic acid molecule comprising one ormore nucleotides, using a method selected from the group consisting ofsolid phase phosphoramidite, solution phase phosphoramidite, solid phaseH-phosphonate, solution phase H-phosphonate, hybrid phasephosphoramidite, and hybrid phase H-phosphonate-based synthetic methods;b) contacting said nucleic acid molecule from step (a) with aqueousalkylamine, a low-volatility amino compound, or combinations thereof,under conditions suitable for the removal of any 2′-amino protectinggroups, exocyclic amino (base) protecting groups and/or phosphateprotecting groups, which may be individually present or absent, fromsaid molecule; c) contacting reaction mixture having said nucleic acidmolecule from step (b) with pyridine-HF, DMAP-HF, urea-HF, TSAF, DAST,polyvinyl pyridine-HF or an amine-HF reagent of formula AA

and a polar solvent under conditions suitable for the removal of a silylprotecting group and/or a 2′-OH protecting group, wherein R¹ is alkyl,aryl, heteroaryl, aralkyl or heteroaralkyl; R² is alkyl, aryl,heteroaryl, aralkyl or heteroaralkyl; R³ is aryl or heteroaryl; and n is1 to 20; d) loading reaction mixture having said nucleic acid moleculefrom step (c) onto an ion exchange chromatography media in a loadingbuffer comprising water, ethanol in about 20 mM sodium phosphate oracetonitrile in about 20 mM sodium phosphate; and e) applying apurification gradient using a suitable elution buffer, analyzing thefractions and allowing for the pure fractions to be pooled and desalted.25. The process of claim 24, further comprising the step of loading saidreaction mixture having said nucleic acid molecule onto a reverse-phasechromatography media in a suitable buffer, prior to or after step d).26. The process of claim 24, further comprising the steps of: annealingsaid nucleic acid molecule with a second nucleic acid molecule to form adouble-stranded nucleic acid molecule, with or without applying thedesalted step in step e); and subjecting the double-stranded nucleicacid molecule to a chromatographic purification.
 27. The process ofclaim 26, wherein the subjecting step comprises: loading said annealeddouble-stranded nucleic acid molecule onto a chromatography media in asuitable buffer; and applying a purification gradient using a suitableelution buffer, analyzing the fractions and allowing for the purefractions to be pooled and desalted.
 28. The process of claim 26,wherein said chromatographic purification is a high-performance liquidchromatography.
 29. The process of claim 26, wherein said nucleic acidmolecule comprises one or more ribonucleotides.
 30. The process of claim26, wherein said double-stranded nucleic acid molecule is an siRNA.